Neurobiology of Aging, Vol. 19, No. 1, pp. 77– 82, 1998
Copyright © 1998 Elsevier Science Inc.
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PII:S0197-4580(97)00166-8

Age-related Changes of Calbindin-D28k,
Calretinin, and Parvalbumin mRNAs in the
Hamster Brain
J. KISHIMOTO,1* T. TSUCHIYA,* H. COX,† P. C. EMSON,† AND Y. NAKAYAMA*
*Life Science Research Laboratories, Shiseido Research Center, 2–12-1 Fukuura, Kanazawa-ku, Yokohama 236, Japan
†MRC Molecular Neuroscience Group, Department of Neurobiology, Babraham Institute, Babraham, Cambridge CB2
4AT, United Kindgom
Received May 28, 1997; Revised October 20, 1997; Accepted November 11, 1997
KISHIMOTO, J., T. TSUCHIYA, H. COX, P. C. EMSON, AND Y. NAKAYAMA. Age-related changes of calbindin-D28k, calretinin,
and parvalbumin mRNAs in the hamster brain. NEUROBIOL AGING 19(1) 77– 82, 1998.—Changes of three different cytosolic Ca2ϩ
binding proteins, calbindin-D28k, calretinin, and parvalbumin mRNA expression in the brain of the hamster during aging were
investigated by in situ hybridization using brains from hamsters aged 4, 9, 13, 19, to 24 months old. In cerebellum area, calbindin-D28k
transcripts showed about 50% to 68% decrease in content in aged-hamster (19 and 24 months old) compared with young (4 months)
and adult (9 months), whereas calretinin and parvalbumin mRNA expression remain unchanged throughout the ages examined.
Calbindin-D28k gene expression was decreased during aging also in the hippocampus (approximately 60% reduction) and striatum
(approximately 25%). In the same areas, striatum and hippocampus, calretinin and parvalbumin mRNA expression in the equivalent
sections were not significantly changed with age. These data raise the possibility that CNS calbindin-D28k expression may be
selectively down-regulated during aging. The statistically significant decrease of calbindin-D28k mRNA in the normal aging process
also suggests and provides further support for the hypothesis that this calcium binding protein may have an important role in neuronal
degeneration. © 1998 Elsevier Science Inc.
In situ hybridization

Calcium binding protein

Calbindin-D28k

IN neurons, impaired Ca2ϩ homeostasis may have a critical role in
cellular aging process both in normal aging and in neurodegenerative conditions. Thus, a sustained increase of free intracellular
Ca2ϩ concentration could affect fundamental aspects of neuronal
function, such as synaptic transmission, maintenance of the cytoskelton, and calcium mediated enzymatic reactions, and could
lead to ultimately to cell death (2).
EF-hand type neuronal calcium binding proteins, such as
calbindin-D28k, calretinin, and parvalbumin, have received much
attention in the last 10 years or so, as one possible function of these
proteins is to act as an intraneuronal calcium buffering proteins (3).
Therefore, the loss of these proteins may result in the disturbance
of intracellular Ca2ϩ concentration (2). Unlike other ubiquitous
and trigger-type calcium binding proteins such as calmodulin,
calbindin-D28k and parvalbumin are expressed in a distinct
sub-set of neurons in the central nervous system (CNS) and several
detailed distribution studies of these proteins have been carried out
in the whole brain area (4). Calretinin, which is highly homologous
at the amino acid sequence level with calbindin-D28k (18),
nevertheless, also shows a distinct and specific distribution pattern
in the brain entirely separate from calbindin-D28k (10,17). Of

Calretinin

Parvalbumin

Aging

Hamster

these, detailed study of the age-related change has been carried out
with calbindin-D28k, and the specific loss of this protein in the
cerebellum during aging have been reported in the mouse (9) and
rat (1). Also, a decrease of calbindin-D28k immunoreactivity has
been reported in the aged rat retina, although no change in
calretinin was reported (15). However, another group has reported
no significant change in both calbindin-D28k and calretinin in the
cerebellum but did show a decrease of these proteins in the
hippocampus of the aging rat (19). Moreover, another group
demonstrated no changes of calbindin-D28k immunoreactivity in
cerebellum tissue and other brain regions in the aging rat using an
immuno-assay system (13). These inconsistent results may be due
to the use of different methods of detection, areas investigated in
the brain, age points tested, and species differences. There has
been so far no study of parvalbumin expression during the aging
process. Thus, further careful studies are needed to obtain a
consensus view as to whether these calcium binding proteins are
regulated during aging.
Thus, the aim of the present study was to investigate the
expression of these three calcium binding proteins during aging,
using in situ hybridization methods, sampling several brain areas,

1
Address correspondence to: Jiro Kishimoto, Life Science Research Laboratories, Shiseido Research Center, 2–12-1 Fukuura, Kanazawa-ku, Yokohama
236, Japan.

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KISHIMOTO ET AL.

and at sequential time points during aging, to build up a clear
picture as to whether expression of these important calcium
buffering proteins is regulated with age.

tial amount of 14-Carbon standard (American Radiolabeled Chemicals, St. Louis, MO), ranged from 8 to 1223 nCi/g weight, were
put on each film at the same time to adjust the different exposure
condition between the films.

METHODS

General
Male Syrian hamsters were obtained at 8 –9 weeks of age and
bred in our animal quarters until they reach the desired age. They
were housed in a 12 h light/12 h dark environment, with lights on
at 0700 h. The temperature was maintained at 23 Ϯ 3°C and
animal food and water were available ad lib. The animals were
assigned to five different age groups as 4, 9, 13, 19, or 24 month
old. Each group was obtained from a breeder at different times of
the year so that the experiments could be performed simultaneously. Each group consisted of at least ten animals at the
beginning of the experiment, although there was some attrition due
to natural causes in the older groups. Animals were decapitated
and the brains were carefully dissected and kept at Ϫ80°C until
use. The study was approved by the Animal Research Committee
of the Shiseido Research Center.
Oligonucleotide Labeling
Antisense oligonucleotides were synthesized for the three
calcium binding protein. These were complementary to the amino
acids 31– 41, 71– 81, 186 –196, 241–250 of calbindin D-28k (14),
and 24 –34, 54 – 63, 79 – 88 of parvalbumin (6). For the calretinin,
the bases 239 –271 of the calretinin cDNA (16) were chosen,
which have no significant homology with calbindin-D28k. Oligonucleotides were 3Ј end-labeled with [35S]dATP using terminal
deoxynucleotidyl transferase (Pharmacia, UK) to a specific activity of Ͼ1 ϫ 107 d.p.m./␮g. The labeled oligonucleotides were
purified by G-50 gel filtration column. The specific radioactivity
of the fraction was monitored by a scintillation counting before
use.
In Situ Hybridization
Radioactive in situ hybridization with 3Ј end labeled oligonucleotide probes was carried out as described previously (12)
Briefly, frozen-blocks of hamster brains were cut into 20-␮m
sections coronally on a cryostat. Three consecutive sets of sequential sections of the striatum, hippocampus, and cerebellum levels in
each hamster brain were hybridized with the probe for calbindinD28k, calretinin, and parvalbumin (i.e., three sections per each
probe by every third section). These are thaw-mounted onto
Vectabond (Vector Lab., Burlingame, USA) coated slides. Sections were fixed with 4% PFA for 30 min, acetylated in 0.25%
acetic anhydride in 0.1 M triethanolamine/0.9% NaCl for 10 min
at room temperature, and dehydrated through graded series of
ethanol. Approximately 5 fmoles/␮L of labeled oligonucleotides
were added in hybridization buffer (50% deionized formamide,
4 ϫ SSC (1ϫ SSC ϭ 0.15 M NaCl/0.015 M sodium citrate), 10%
dextran sulfate, 1 ϫ Denhardt’s solution, 400 ␮g/mL sonicated
salmon sperm DNA, 3% mercaptoethanol). In this hybridization
mixture, Each of three consecutive sections were incubated at
37°C for 16 –18 h with the probe for calbindin-D28k, calretinin,
and parvalbumin, respectively. The hybridized sections were
washed in 1 ϫ SSC three times at 55°C and once at room
temperature, then dehydrated sequentially in 70 and 95% ethanol.
To assess the amount of each calcium binding protein mRNA as a
radioactivity unit, slides were exposed to large Hyperfilm-␤max
autoradiography film (Amersham, Arlington Heights, USA) for 1
week (for calbindin-D28K) or 4 weeks (for calretinin and parvalbumin). Brain paste sections (20 ␮m), including series of sequen-

Control Experiments
Some sections were hybridized with labeled oligonucleotides in
the presence of excess unlabelled same oligonucleotides at a
concentration of 1000 fmoles/␮L (200 times higher than [a-35S]
labeled oligonucleotides) to check the specificity of the in situ
signal.
Image Analysis
To obtain reliable and comparable in situ signals on the
autoradiography image, we chose relatively uniform areas or
nuclei in the sections for analysis. Gray scale autoradiographic
images of mRNA signals on the section were obtained by scanning
films with an Image Scanner (Epson, Nagano, Japan), and the
radioactivity per 1.0 mm2 of each section on autoradiographic film
was calculated by NIH Image 1.55 analysis program using
threshold option with radioactive standard. Thus, firstly the appropriate threshold value was chose in order to distinguish specific
signals from background, and measure the “positive” area of 1.0
mm2 at five points at random per area (e.g., cerebellum) in each
section, then analyze to obtain the mean value. Integrated density
was then obtained after background level was subtracted from
mean value. Averaged values pooled from three sections (i.e.,
fifteen points) were used for the value for that animal. The amount
of radioactivity was then interpreted from the value of the brain
paste section containing carbon-14 standard put on the same film.
Statistical Analysis
The amount of radioactivity in all experiments were analyzed
statistically. Multiple comparison between the different age groups
were analyzed by one-way analysis of variance (one-way
ANOVA) followed by Scheffe’s test. Values represent means Ϯ
SD. All probabilities presented are 2-tailed. Differences were
considered significant when p Ͻ 0.05.
RESULTS

Changes of mRNA Expression for Calcium Binding Proteins in
the Cerebellum with Aging
Of sections we tested in this study, the cerebellum was the only
area which exhibited measurable in situ signals for all three
calcium binding proteins. Figure 1 shows the autoradiographic
film image of in situ signals for calbindin-D28k, calretinin, and
parvalbumin in the cerebellum at different age. The film image for
calbindin indicated the expression was limited only in Purkinje
cells and the broader signal for calretinin indicated this was
localized in the granule cell layer. Parvalbumin was expressed both
in the Purkinje cells and in the molecular cell layer, but not in the
granule cell layer. The amount of radioactivity per area on the
sections for these protein transcripts is also shown in Figure 2 and
was calculated from the radio-isotope standards as described in
Methods.
In the cerebellum, a significant (F (4, 27) ϭ 9.96, p Ͻ 0.001)
decrease of calbindin D-28k mRNA expression with aging was
observed. Thus, there was no change between young (4 months;
33.1 Ϯ 10.6 nCi/g tissue) and adult (9 months; 32.0 Ϯ 6.0 nCi/g
tissue). The 13-month-old group (18.4 Ϯ 8.3 nCi/g tissue) showed
a decline, but it was not significant. This difference reached a
significance (p Ͻ 0.05) at 19 months (17.5 Ϯ 7.9 nCi/g tissue),
approximately a 50% reduction as compared with young and adult

Ca2ϩ BINDING PROTEINS IN AGED BRAIN

79

FIG. 1. Autoradiography film images of calbindin-D28k, calretinin, and parvalbumin in situ signals in cerebellum sections with different ages indicated.

age groups (Fig. 2). The expression was further decreased at 24
months (10.5 Ϯ 5.4 nCi/g tissue; approximately a 68% reduction
from young and adult age groups) with significance (p Ͻ 0.01).
On the other hand, parvalbumin mRNA expression in the
cerebellum were entirely unchanged (F (4, 27) ϭ 1.77, p ϭ 0.16)
throughout aging from 4 to 24 months (Fig. 2).
Regarding calretinin mRNA expression, although the overall
change in the cerebellum with aging was significant (F (4, 27) ϭ
2.92, p ϭ 0.04), none of the individual comparisons reached
significance; particularly, values at old age groups (19 and 24
months) failed to show a clear decrease compared with young and
adult age groups (Fig. 2).
Control experiments with excess unlabelled probe abolished all
specific in situ signals on the films.
Changes of mRNA Expression for Calcium Binding Proteins in
Other Brain Areas with Aging
Other than the cerebellum area,, strong signals were obtained
from the hippocampus and caudate-putamen (striatum) for calbindin-D28k, some nuclei of the thalamus for calretinin, and the
reticular thalamic nucleus for parvalbumin. Thus, further analysis
was focused on these areas for each of the respective calcium
binding protein transcripts.
The area of the dentate gyrus in the hippocampus showed a
significant (F (4, 28) ϭ 19.86, p Ͻ 0.001) age-related decrease in
mRNA expression for calbindin-D28k as similarly with the cerebellum (Figs. 3A, 4A). The magnitude of decline was also similar
to that in the cerebellum; an approximately 55 and 65% decline
was observed at 19 months (18.4 Ϯ 8.3 nCi/g tissue) and 24
months (14.1 Ϯ 2.4 nCi/g tissue; Fig. 4A) respectively, as
compared with 4 months (40.9 Ϯ 5.9 nCi/g tissue).

FIG. 2. Expression levels of mRNA for three calcium binding proteins,
calbindin-D28k (Ⅺ), calretinin (‚), and parvalbumin (F) in the cerebellum. The results shown are calculated from the radioactivity standard
described in Methods. Multiple comparisons between the different age
groups were statistically analyzed by Scheffe’s test; values that are
significantly different are denoted with the same letter designation (capital
letter: p Ͻ 0.01, small letter: p Ͻ 0.05). Each value indicates mean Ϯ SD
for 5– 8 animals.

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KISHIMOTO ET AL.

FIG. 3. Autoradiography film images of three calcium binding proteins in situ signals in the: (A) calbindin-D28k in the hippocampus; (B) calbindin-D28k
in the striatum; (C), calretinin in the nucleus of the thalamus; and (D), parvalbumin in the reticular thalamic nucleus. In each figure, the left image shows
a representative section of a 4-month-old, and the right shows that of a 24-month-old.

The striatum region also showed a significant (F (4, 28) ϭ 9.31,
p Ͻ 0.01) age-related decrease of calbindin-D28k mRNA expression, but the decrease showed significance only when aged groups
(13 months (26.9 Ϯ 5.0 nCi/g tissue), 19 months (27.4 Ϯ 2.2 nCi/g
tissue), and 24 months (26.9 Ϯ 3.1 nCi/g tissue)) were compared
with the 4-month-old group (36.9 Ϯ 4.3 nCi/g tissue), and the
reduction rates were only 25%, which is less than that observed in
hippocampus (Figs. 3B, 4B).
Although overall changes of expression of calretinin mRNA in
the thalamus with aging was significant (F (4, 28) ϭ 3.17, p ϭ
0.03) similar to the case in cerebellum, any comparison between
individual groups failed to show a significance, indicating no
obvious relation between the aging process and the change of
calretinin mRNA expression in this area (Figs. 3C and 4C).
Parvalbumin mRNA expression in the reticular thalamic nucleus, which is one of the strongest sites for parvalbumin expression in the brain, was entirely unchanged (F (4, 27) ϭ 1.42, p ϭ
0.25) throughout the age groups examined in the present study
(Figs. 3D and 4D).
DISCUSSION

In the present study, we analyzed the age-related changes of in
situ mRNA signals for three neuronal calcium binding proteins,
calbindin-D28k, calretinin, and parvalbumin, in the brain of
the hamster.
We used the hamster because this study was done as a part of
an ongoing aging study using this rodent species. Although
detailed distribution studies of these calcium binding proteins for
rodent species have been performed in the rat (4,17), a striking
conservation in the distribution of most positive cell types has been
reported among rodents, monkeys, and humans in the hippocampus, striatum, and cerebellum (2). In the present study, the
distribution pattern of these proteins in the cerebellum in the

hamster is basically identical to that of reported in the rat using the
same in situ hybridization technique (11), that is; strong and
specific expression of calbindin-D28k in Purkinje cells, calretinin
in granule cells, and parvalbumin in the Purkinje cells and the
molecular layer. Furthermore strong expression of calbindin-D28k
in the dentate gyrus of hippocampus, calretinin in the some nuclei
of the thalamus, and parvalbumin in the reticular thalamic nucleus
are all consistent with previous observations in the rat (4,10,17),
implying a well conserved distribution pattern for these calcium
binding proteins between rats and hamsters and among rodent
species.
As our analytical tool, we chose to use radioactive in situ
hybridization technique and film autoradiography. This technique
has a number of advantages over other immunochemical/histochemical methods. Thus there is direct relationship between the
strength of the film signal and the tissue mRNA content because
there is no peroxidase or avidin biotin amplification step during the
reaction. Further, the use of radioactive standard on each film
makes it possible to compare absolute amount of radioactivity
between the sections. One disadvantage of the film analysis used
here is that it is not always easy to see which cell type is affected.
However the cerebellum distribution of calcium binding protein
mRNAs is so characteristic and specific that the loss of calbindinD28k mRNA is clearly localized to the Purkinje cells.
Of three proteins examined here, only calbindin-D28k showed
a selective decrease in mRNA expression level in aging, whereas
of the other two calcium binding proteins, parvalbumin mRNA
was unchanged, and the levels of calretinin mRNA failed to show
clear age-related changes in the area of hamster brains examined in
the present study. Selective age related loss of calbindin-D28k in
the cerebellum has been reported in the rat (1,8) and mouse (9).
Also a decrease of this protein in the rat hippocampus was reported
using immunohistochemical detection methods (19). Although this

Ca2ϩ BINDING PROTEINS IN AGED BRAIN

81

FIG. 4. Expression levels of three calcium binding proteins in the: (A) calbindin-D28k in the hippocampus; (B) calbindin-D28k in the striatum;
(C) calretinin in the nucleus of the thalamus; and (D) parvalbumin in the reticular thalamic nucleus. Multiple comparisons between the different
age groups were statistically analyzed by Scheffe’s test; values that are significantly different are denoted with the same letter designation
(capital letter: p Ͻ 0.01, small letter: p Ͻ 0.05). Each value indicates mean Ϯ SD for 5– 8 animals.

group only measured calbindin-D28k immunoreactivity using
whole hippocampus/cortex tissue by western blotting, a more
recent study showed a marked decrease of calbindin-D28k protein
in the dentate gyrus within the “aged” hippocampus both in rat and
rabbit species (5), consistent with our present observations on the
hippocampus. Selective loss of calbindin-D28k protein was also
reported in the rat retina (15).
There have been a few studies so far about the age-related
changes of calretinin in the brain. Among them, no decrease of

calretinin has been reported in the rat retina (15) and cerebellum
(19). The latter group also examined calretinin immuno-reactivity
in the hippocampal cortex homogenate and found a significant
decrease. There has been only one recent report investigating
age-related changes in parvalbumin, which showed no significant
change of parvalbumin immunoreactivity in the hippocampus
region of rat and rabbit (5) consistent with our results on
parvalbumin expression in the cerebellum and reticular thalamic
nucleus.

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KISHIMOTO ET AL.

Although there has been no clear consensus conclusion about
age-related changes of these calcium binding proteins in the CNS,
the majority of previous data and our present study suggests that
calbindin-D28k is the most sensitive neuronal calcium binding
protein among three calcium binding proteins examined in terms
of aging, calretinin may show some regional sensitivity, and
parvalbumin would seem to be relatively stable during the normal
aging process.
Down-regulation of calbindin-D28k gene expression or loss of
the sub-population of neurons that express the gene for this protein
seem to occur not only in the particular region or specific nucleus
but throughout the most of the brain regions as we could see on the
film image. For example, on sections examined for hippocampal
expression, the calbindin-D28k mRNA signal was also clearly
reduced due to aging not only in the dentate gyrus of the
hippocampus, the area examined in detail, but also in most of the
other areas on the film, including the cells of the basal nucleus of
Meynert and stria medullaris of the thalamus (see Fig. 4A). Any
possibility that this could be an artifact due to in situ procedure
could be eliminated as the probes for other two calcium binding
proteins that showed basically unchanged film images during
aging were processed at the same time as the sections used to
visualize the calbindin-D28k mRNAs
The decrease of calbindin-D28K mRNA observed here in the

aged hamster is consistent with an important role for this calcium
binding protein as a buffering protein maintaining intracellular
Ca2ϩ homeostasis in neuronal cells and a decline in expression
contributing to aged related decline in neuronal function (2). This
also raises the related question as to whether the gradually
decrease of calbindin-D28k expression during the long-term aging
process may be accelerated in neurodegenerative disease such as
Alzheimer’s disease, or epilepsy and ischemia (7). This may be
investigated using animal models of these neurodegenerative
disease.
Our present study shows that the in situ hybridization approach
is a very useful tool for the assessment of the changes of the gene
expression during the aging process in the brain. Further detailed
studies on age-related changes of these calcium binding proteins in
several species including humans could enhance our understanding
of the role of these potentially important, but functionally still little
known proteins in the CNS during normal and pathological aging
process.
ACKNOWLEDGEMENTS

The authors express their gratitude to Mrs. K. Westmore for her
technical assistance in in situ hybridization work, and Ms. Y. Takahashi
and Ms. Y. Kaminaka for image analysis.

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