- Email: [email protected]
Age-dependent and region-specific alteration of parvalbumin neurons and perineuronal nets in the mouse cerebral cortex
Accepted Manuscript Age-dependent and region-specific alteration of parvalbumin neurons and perineuronal nets in the mouse cerebral cortex Hiroshi Ueno, Keizo Takao, Shunsuke Suemitsu, Shinji Murakami, Naoya Kitamura, Kenta Wani, Motoi Okamoto, Shozo Aoki, Takeshi Ishihara PII:
S0197-0186(17)30349-2
DOI:
10.1016/j.neuint.2017.11.001
Reference:
NCI 4160
To appear in:
Neurochemistry International
Received Date: 14 June 2017 Revised Date:
28 October 2017
Accepted Date: 1 November 2017
Please cite this article as: Ueno, H., Takao, K., Suemitsu, S., Murakami, S., Kitamura, N., Wani, K., Okamoto, M., Aoki, S., Ishihara, T., Age-dependent and region-specific alteration of parvalbumin neurons and perineuronal nets in the mouse cerebral cortex, Neurochemistry International (2017), doi: 10.1016/j.neuint.2017.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
Research Article
Age-Dependent and Region-Specific Alteration of Parvalbumin
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Neurons and Perineuronal Nets in the Mouse Cerebral Cortex1
Hiroshi Uenoa, b, *, Keizo Takaoc, Shunsuke Suemitsud, Shinji Murakamid,
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Naoya Kitamurad, Kenta Wanid, Motoi Okamotob, Shozo Aokid, Takeshi Ishiharad
a
1
Department of Medical Technology, Kawasaki University of Medical Welfare,
Abbreviations: FrA, frontal association cortex; DLO, dorsolateral orbital cortex; LO, lateral orbital cortex;
VO, ventral orbital cortex; Cg, cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; DP, dorsal
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peduncular cortex; M1, primary motor cortex; M2, secondary motor cortex; MPtA, medial parietal
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association cortex; LPtA, lateral parietal association cortex; PTPR, parietal cortex, post, rostral; S1Tr, primary somatosensory cortex–trunk region; S1BF, primary somatosensory cortex–barrel field; S2,
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secondary somatosensory cortex; V2MM, secondary visual cortex–mediomedial area; V2ML, secondary visual cortex, mediolateral area; V1M, primary visual cortex, monocular area; V1B, primary visual cortex,
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binocular area; V2L, secondary visual cortex, lateral area; Au1, primary auditory cortex; AuD, secondary auditory cortex, dorsal area; AuV, secondary auditory cortex, ventral area; TeA, temporal association cortex; Ect, ectorhinal cortex; PRh, perirhinal cortex; DIEnt, dorsintermed entorhinal cortex; DLEnt, dorsolateral entorhinal cortex; RSD, retrosplenial dysgranular cortex; RSGc, retrosplenial granular cortex, c region; RSGb, retrosplenial granular cortex, b region; RSGa, retrosplenial granular cortex, a region; AD, Alzheimer’s Disease.
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ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
Okayama, 701-0193, Japan b
Department of Medical Technology, Graduate School of Health Sciences,
c
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Okayama University, Okayama, 700-8558, Japan Life Science Research Center, University of Toyama, Toyama, 930-0194, Japan
Department of Psychiatry, Kawasaki Medical School, Kurashiki, 701-0192,
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d
Japan
Author Information
Hiroshi Ueno, E-mail: [email protected] Keizo Takao, E-mail: [email protected]
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Shunsuke Suemitsu, E-mail: [email protected]
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Shinji Murakami, E-mail: [email protected] Naoya Kitamura, E-mail: [email protected]
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Kenta Wani, E-mail: [email protected] Motoi Okamoto, E-mail: [email protected]
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Shozo Aoki, E-mail: [email protected] Takeshi Ishihara, E-mail: [email protected]
*Corresponding author. Hiroshi Ueno, PhD. Address: Department of Medical Technology, Kawasaki University of Medical
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ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
Welfare, 288, Matsushima, Kurashiki, Okayama, 701-0193, Japan Phone: +81-86-462-1111, Fax: +81-86-462-1193
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E-mail address: [email protected] (H. Ueno)
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ABSTRACT Cognitive function declines with age. Such function depends on in
the
frontal
cortex.
Pyramidal
neurons,
and
the
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-oscillation
parvalbumin-expressing interneurons (PV neurons) that control them, are important for the generation of -oscillation. The mechanism by which
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cognitive function declines is unclear. Perineuronal nets (PNNs) mainly surround the soma and proximal dendrites and axon segments of PV neurons in the cerebral cortex. Previous evidence indicates that PNNs inhibit neural plasticity. If this is true, an increase in the number of neurons surrounded by PNNs or in the thickness or density of the PNNs around neurons could decrease plasticity in the cortex. To determine if an aging-related change in cortical PNNs occurs, we examined the influence aging
on
PV
neurons
and
whether
Wisteria
floribunda
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of
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agglutinin-positive PNNs differ depending on the cortical area. The results showed that the number of PV neurons/mm2 did not change in many areas
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of the cortex as mice aged. In contrast, the number of neurons in the sensory cortex surrounded by PNNs increased as mice aged. Thus, with
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age, PNN density increases in some cortical areas but not in others. In addition, the expression level of PV protein in PV neurons decreased with aging in the whole cortex. We suggest that decreased expression of PV protein impairs fast spiking in PV neurons. We propose that PNNs surround more neurons as age increases. This aging-related increase in PNNs decreases plasticity in the cerebral cortex and reduces cognitive function. The first step in investigating this proposal would be to
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determine if PNN density increases with age.
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Keywords: aging, cerebral cortex, mouse, parvalbumin, perineuronal nets
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ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
1. Introduction As animals naturally age, their cognitive function declines; however, the
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underlying mechanisms driving this process have not been elucidated. No one has clarified yet whether aging-related change occurs in the whole brain or in
localized brain regions. Cognitive function in the brain depends on γ-oscillation, in
turn
depends on
the
function
of
pyramidal neurons and
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which
parvalbumin-expressing interneurons (PV neurons (Sohal et al., 2009; Kleschevnikov et al., 2012; Lewis et al., 2005).
PV neurons are one category of GABAergic interneurons among several categories. We can distinguish different types of GABAergic neurons by noting which calcium-binding protein (CaBP) they express: parvalbumin, calbindin, or calretinin (Markram et al., 2004). Categorizing interneurons according to their
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calcium-binding protein is useful because these neurons exhibit a wide variety of
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other attributes such as morphology, physiology, and the expression of other molecules. The type of GABAergic interneuron that expresses parvalbumin
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accounts for about half of all GABAergic interneurons in the cerebral cortex (Rudy et al., 2011). A single PV neuron makes synapses with hundreds of
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pyramidal neurons and controls the synchronous firing of these neurons (Kawaguchi, 2001). This is essential for the generation of -oscillations related to working memory (Bartos et al., 2007; Sohal et al., 2009). Therefore, a decrease in PV protein level leads to an impairment in the frequent firing of PV neurons and a loss of -oscillatory activity (Sohal et al., 2009; Volman et al., 2011). Previous work suggests that PV neurons cause functional deterioration with age and accelerate cognitive impairment (Lewis, 2012; Takahasi, 2002).
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In the cortex, approximately 80% of PV neurons are surrounded by extracellular matrix molecules (Berretta et al., 2015). These molecules form a
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mesh-like structure, called a perineuronal net (PNN). PNNs surround the cell bodies, proximal dendrites, and initial axon segments of PV neurons. They are mainly composed of chondroitin sulfate proteoglycans (CSPGs), hyaluronic acid,
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and link proteins (Bandtlow and Zimmermann, 2000; Dityatev and Schachner, 2003). CSPGs expressed in the central nervous system include lectican, aggrecan, brevican, and neurocan. PNNs
mainly
function
in
controlling
synaptic
plasticity
and
in
neuroprotection (Wang and Fawcett, 2012). They appear during development, near the end of the critical period, in the sensory cortex (Chevaleyre and Piskorowski, 2014; Sun, 2009). Dissolving PNNs with chondroitinase ABC in the
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primary visual cortex of adult rats induced experience-driven plasticity, similar to
2002).
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the plasticity observed in young rats during the critical period (Pizzorusso et al.,
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Many studies have indicated that the brain exhibits a decrease in plasticity with age (Gray and Barnes, 2015; Mora, 2013; Lehmann et al., 2012). If PNNs
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do indeed inhibit plasticity, then an aging-related increase in the number of neurons surrounded by PNN would reduce plasticity, and maybe cognitive function, in older individuals. In this study, we looked for age-related increases in the number of PNNs in the cerebral cortex to determine if such changes are correlated with cognitive decline.
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2. Materials and methods 2.1. Animals
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We used male mice (C57BL/6) for the experiments. The day of birth was designated as postnatal day 0. We divided the mice into three groups according
to age: 2 months postnatally (2 months, n =5), 6 months postnatally (6 months, n
= 5), and 12 months postnatally (12 months, n = 5). We purchased the animals
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from Charles River Laboratories (Kanagawa, Japan) and housed them in cages (3–5 animals per cage) with food and water provided ad libitum under a 12-h light/dark cycle at 23°C–26°C. We made every effort to minimize the number of animals used and their suffering. These experiments complied with the U.S. National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised in 1996) and were approved by the
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Committee for Animal Experiments at Kawasaki Medical School Advanced
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Research Center.
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2.2. Tissue preparation
For tissue preparation from mice (2, 6, and 12 months old), we deeply
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anesthetized the animals with a lethal dose of sodium pentobarbital (120 mg/kg, i.p.) and transcardially perfused them with ice-cold phosphate-buffered saline (PBS) for 2 min and then 4% paraformaldehyde in PBS (pH 7.4) for 10 min (10 ml/min). In all cases, we dissected all the brains and post-fixed them overnight with 4% paraformaldehyde in PBS at 4°C and cryoprotected them by immersion in 15% sucrose for 12 h followed by 30% sucrose for 20 h at 4°C. To cut sections, we froze the brains in O.C.T. Compound (Tissue-Tek; Sakuma Finetek, Tokyo,
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Japan) using dry ice-cold normal hexane and we prepared serial coronal sections of 40-µm thickness using a cryostat (CM3050S; Leica Wetzlar,
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Germany) at -20°C. We collected sections in ice-cold PBS containing 0.05% sodium azide.
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2.3. Immunohistochemistry
We visualized PNNs with antibodies that recognize sugar chains. The lectins Wisteria floribunda agglutinin (WFA) and Vicia villosa agglutinin bind to the terminal N-acetylgalactosamine residues of CSPGs (Young and Williams, 1985). Lectin WFA has been frequently used as a comprehensive marker of PNNs. We treated the cryostat sections with 0.1% Triton X-100 in PBS at room temperature for 15 min. After three washes in PBS, we incubated the sections with 10%
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normal goat serum (ImmunoBioScience Corp., Mukilteo, WA) in PBS at room
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temperature for 1 h, we washed them three times in PBS, and incubated them overnight at 4°C in PBS containing biotinylated WFA (B-1355, Vector
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Laboratories, Funakoshi Co., Tokyo, Japan; 1:200) and primary antibodies (described below). After washing in PBS, we incubated the sections with secondary
antibodies
(indicated
below)
and
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corresponding
streptavidin-conjugated Texas Red (SA-5006; Vector Laboratories) at room temperature for 2 h. We rinsed the labeled sections again with PBS and we mounted them on glass slides with Vectashield medium (H-1400; Vector Laboratories). We stored the prepared slides at 4°C until we used them in the microscopy analysis.
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2.4. Antibodies We used the following primary antibodies for staining: mouse anti-parvalbumin
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(clone PARV-19, P3088; Sigma-Aldrich Japan, Tokyo, Japan; 1:1,000), rabbit anti-calbindin (ab25085; Abcam, Tokyo, Japan; 1:400), mouse anti-calretinin (clone
6B8.2,
MAB1568;
Millipore,
Tokyo,
Japan;
1:500),
rabbit
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anti-somatostatin (T-4103; Peninsula Laboratories, Belmont, CA; 1:2,000),
rabbit anti-vasoactive-intestinal peptide (20077; Immunostar, Hudson, WI; 1:1,000), mouse anti-GAD67 (clone 1G10.2, MAB5406; Millipore; 1:1,000), and mouse anti-NeuN (clone A60, MAB377; Millipore; 1:500). We used the following secondary antibodies for visualization: Alexa Fluor 488-conjugated goat anti-mouse IgG (ab150113; Abcam; 1:1,000) and Alexa Fluor 488-conjugated
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2.5. Microscopy imaging
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goat anti-rabbit IgG (ab150077; Abcam; 1:1,000).
For quantification of the number and fluorescence intensity of PV- and
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WFA-positive neurons, we used confocal laser scanning microscopy to obtain images of stained sections. Images (1024 × 1024 pixels) were saved as TIFF
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files using ZEN software. Briefly, we performed the analysis was performed using a × 10 or × 20 objective lens and a pinhole setting that corresponded to a focal plane thickness of less than 1 m. Images from whole sections using a × 10 objective lens of the fluorescence microscope (BZ-X; KEYENCE, Tokyo, Japan) and we merged them using KEYENCE BZ-X Analyzer software (KEYENCE).
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2.6. Quantification of labeled neurons In this experiment, we measured three attributes of labeled neurons in the
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cortices of the mice. 1) The density of labeled neurons. For this, we counted the number of labeled neurons in a cortical region of the cortex (defined by the mouse atlas; Paxinos and Franklin, 2012) and we measured the area of that
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region. We then calculated labeled neurons/mm 2. 2) The distribution of labeled
neurons. For this, we examined the distribution across the different cortical regions that we studied. 3) The fluorescence intensity of labeled neurons. For this, we selected 12 sections from each mouse brain and stained them as described above. On 8-bit images of each section, we manually outlined each PV-positive neuron cell body and measured the gray level with NIH ImageJ (NIH, Bethesda, MD; http://rsb.info.nih.gov/nih-image/). Prior to capture, the exposure
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time, gain, and offset were carefully set to ensure a strong signal but to avoid
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saturation. Identical capture conditions were used for all sections. Background intensity was subtracted using unstained portions of each section. We acquired
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all confocal images as TIFF files and analyzed them with NIH ImageJ. We coded the slides and a blinded independent observer quantified them. We performed
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estimations of neuronal density (cells/mm2).
2.7. Data analysis
Data are expressed as mean ± S.E.M. Statistical significance was determined using one-way ANOVA with Bonferroni multiple comparison test, and was set at *p < 0.05.
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3. Results 3.1. Effect of aging on PV neurons and WFA-positive PNNs in the mouse
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cerebral cortex As shown in Figures 1A and 2 (green) the fluorescence intensity of PV neurons was lower in 12-month-old mice. As shown in Figures 1B and 2 (red),
the laminar distribution of WFA-positive PNNs throughout the cortical layers was
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very similar in 2-month- and 12-month-old mice. Weaker PV immunoreactivity was observed in all 12-month-old mouse cortices examined, compared to 2-month-old mouse cortices.
3.2. Effect of aging on PV neuron density and WFA-positive PNN density in
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the mouse cerebral cortex
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Figure 3A shows the density of PV neurons in each of the cortical areas that we examined and for each age. In most of the 33 areas that we examined, there
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was no significant difference between the density of PV neurons at different ages. In three areas, Cg/M2. AuD, and RSGb, the density of PV neurons was
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significantly higher in 12-month-old mice than in 2-month-old mice. Figure 3B shows the density of WFA-positive PNNs in each of the cortical
areas that we examined and for each age. In some of the areas that we examined, there was no significant difference between the density of WFA-positive PNNs at different ages. In other areas, however, the density in 12-month-old mice was significantly higher than that in 2-month-old mice. These regions were: frontal areas: VO, Cg, M2; parietal areas: MPtA, S1BF; occipital
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areas: V2MM, V1B; auditory temporal cortex: Au1, AuD, AuV; other temporal
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cortex: TeA, DIEnt; retrosplenial cortex: RSGb.
3.3. Effect of aging on the co-localization of PV neurons and WFA-positive PNNs in the mouse cerebral cortex
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As Figure 4A shows, there was no significant difference in this percentage
between 2-month- and 12-month-old mice in most of the areas that we examined. The percentage was significantly higher in 12-month-old mice in the following four areas: frontal - Cg; parietal - S1Tr; occipital - V2MM; auditory temporal AuD.
Figure 4B shows the percentage of WFA-positive PNNs that covered PV neurons in each of the cortical areas that we examined and for each age. In
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some of the areas that we examined, there was no significant difference
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between the percentage of WFA-positive PNNs that covered PV neurons at different ages. In other areas, however, the percentage in 12-month-old mice
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was significantly lower than that in 2-month-old mice. These regions were: parietal areas: S1BF, S2; occipital areas: V2MM, V1B, V2L; auditory temporal
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cortex: Au1, AuD; retrosplenial cortex: RSD, RSGb. Newly formed WFA-positive PNNs did not cover PV neurons in these areas in 12-month-old mice.
3.4. Effect of aging on the fluorescence intensity of PV neurons in the mouse cerebral cortex To examine the effect of aging on the expression of PV protein in the brain, we analyzed the fluorescence intensity of each PV-positive neuronal soma in
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each of the mouse cortical areas we examined. As shown in Figure 5, the fluorescence intensity of PV neurons was significantly lower in 12-month-old
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mice than in 2-month-old mice. Figure 5 also shows that the intensity of PV labeling differed in each of the cortical areas that we examined and for each age.
These results raise the possibility that the possibility that PV neuron function
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differs significantly between 2 months and 12 months of age in mice.
3.5. Effect of aging on the soma area of PV neurons in the mouse cerebral cortex
The area of the soma in PV-labeled neurons did not significantly differ between 2-month-old and 12-month-old mice in 23 of the 33 brain areas that we examined. In 10 brain areas, the size of each PV-positive soma was smaller in
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12-month-old mice than in 2-month-old mice. These 10 areas were: frontal: Vo,
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M2; parietal: S2; occipital: V2MM, V1M, V2L; auditory temporal: AuD; other
3.6.
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temporal: DLEnt; retrosplenial: RSGa, RSGb.
Co-localization
of
newly
formed
WFA-positive
PNNs
and
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GAD67-expressing neurons in the mouse primary somatosensory cortex and primary auditory cortex with aging In both the primary somatosensory cortex (S1BF) and primary auditory
cortex (Au1) of 12-month-old mice, WFA-positive PNNs formed around neurons that were not PV neurons (Fig. 2A–B, 4). To further characterize the neurons surrounded by WFA-positive PNNs in areas S1BF and Au1 of 12-month-old mice, we stained for calbindin (CB), calretinin (CR), somatostatin (SOM), and
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vasoactive-intestinal peptide (VIP) (Fig. 7A–D, 8A–D). In both the primary somatosensory cortex and primary auditory cortex, CB, CR, SOM, and VIP
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neurons were not surrounded by WFA-positive PNNs from the 6-month to the 12-month stages (Fig. 7A–D, 8A–D). Next, we labeled GABAergic neurons
using the anti-GAD67 antibody (Kaufman et al., 1991). In both the primary
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somatosensory cortex and primary auditory cortex of 12-month-old mice, we
observed that some of the WFA-positive PNNs did not contain GAD67 (Fig. 7E, 8E). In addition, we examined which cells surrounded by WFA-positive PNNs that are formed in the adult brain co-localized with NeuN. NeuN was used to identify neurons to exclude non-neuronal cells. Most cells enveloped by WFA-positive PNNs in the 12-month-old primary sensory cortex did not contain NeuN (Fig. 7F, 8F). In some brain regions, some groups of neurons do not
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express NeuN (Mullen et al., 1992, Weyer and Schilling, 2003). In the
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cerebellum, these neurons coincide with GABAergic interneurons (Weyer and Schilling, 2003). According to the distribution and shape of the neurons, the
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findings suggested that the neurons formed by WFA-positive PNNs in the adult primary sensory cortices are GABAergic interneurons, which do not contain
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GAD67.
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4. Discussion This study showed that the influence of aging on PV neurons and
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WFA-positive PNNs differs depending on the cortical area. The number of PNN-positive neurons increased more with age in the primary sensory cortices
than in other cortical areas. Aging-related increases in the percentage of PV
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neurons that are surrounded by PNNs in the association cortices, such as in the
frontal and entorhinal cortices, were not observed. The PV neuron density also did not change in numerous cortical areas. The expression of PV protein was significantly reduced in the aged cortices examined in this study compared to the cortices of 2-month-old mice.
Cognitive impairment with aging is caused by decreases in the numbers of neurons and dendrites in a smaller and more limited area (Burke and Barnes,
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2006). In line with previous findings that cognitive function and memory decline
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with age although neuronal numbers are preserved (Rapp et al., 2002; Freeman et al., 2008; Burke and Barnes, 2006) and that PV neuron density did not
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decrease with aging in the sensory cortices (Karetko-Sysa et al., 2014), we found that PV neuron density remained unchanged with aging in numerous
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cortical areas.
Intracellular Ca2+ greatly influences the physiological, chemical, and
biological processes associated with neurons (Foster, 2007). Many studies have shown that Ca2+ conductance increases with age (Farajnia et al., 2015). It has also been reported that Ca2+ homeostasis is disrupted in the prefrontal cortex of aged monkeys (Chang et al., 2005). PV, a Ca2+-binding protein, is important for regulating intracellular Ca2+ kinetics in PV-expressing neurons (Chard et al,
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1993; Baimbridge et al., 1992). Moreover, a previous study indicated that Ca2+ signaling in PV neurons decreased with age in mice (Jessen et al., 2017), but
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there has been no report of decreased PV protein expression in aged mice. Reduction of PV expression reduces the high-frequency firing of PV neurons
and leads to the loss of -oscillation by PV neurons (Lewis, 2012; Murray et al.,
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2015).
Decreases in sensory input are partially responsible for aging-related decline in PV expression, as PV protein expression in sensory cortical PV neurons depends on sensory input (Patz et al., 2004) and mice show less movement and social behavior with aging (Shoji et al., 2016). Another possible reason may be the aging-dependent decline in brain-derived neurotrophic factor (BDNF) expression in the central nervous system (Adlard et al., 2005; Gooney et
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al., 2004), as BDNF is essential for the expression of PV mRNA in PV neurons
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during development (Huang et al., 1999; Zheng et al., 2011). In line with the results of a previous study (Peinado et al., 1997), we showed
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that the soma areas of PV neurons shrink in numerous cortical areas with aging. In other words, the aging-dependent soma shrinking in PV neurons may reflect
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functional deterioration.
For the first time, we showed that the aging-related increase in
WFA-positive PNN density is region-selective, occurring in the somatosensory, visual, and auditory cortices, rather than in the association cortex. We did not observe aging-related increase in WFA-positive PNN density in areas requiring high flexibility throughout the lifespan, such as the frontal cortex, and we suggest that the plasticity of the association cortex is preserved with aging.
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It remains unclear which types of neurons WFA-positive PNNs surround with aging. In this study, it was shown that these neurons are not positive for CB,
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CR, SOM, VIP, GAD67, and NeuN; a previous study suggested that they may be GAD67-negative GABAergic interneurons (Karetko-Sysa et al., 2014). Based on their morphology,
size,
and
distribution,
the
neurons
surrounded
by
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newly-formed, aging-related, WFA-positive PNNs may be PV-negative PV
neurons or PV neurons expressing PV at levels lower than the detection threshold. Further studies are needed to determine which neurons are surrounded by newly-formed, aging-related, WFA-positive PNNs.
PNN function in protecting neurons from oxidative stress, tau protein toxicity, and glutamic acid excitotoxicity is well documented (Okamoto et al., 1994; Morawski et al., 2010; Morawski et al., 2014; Suttkus et al., 2014 ). We found
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that there was no aging-related increase in PV neurons surrounded by PNNs in
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the association cortex. Furthermore, PV protein expression, which prevents excessive Ca2+ intracellular influx, was found to decrease with aging. A recent
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study found that -oscillation involving PV neurons in the entorhinal cortex was reduced at the early stage of Alzheimer’s disease (AD) (Klein et al., 2016).
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Moreover, reactive oxygen species increase in the brain with aging (Balbi et al., 2015; Zlokovic, 2011), which may promote dementia (de la Torre and Stefano, 2000). The present results indicate that the association cortex, which contains numerous PV neurons without neuroprotective PNNs, may be particularly susceptible to oxidative stress and dementia-related damages with aging; suggesting that there is region-selective vulnerability in the cortex.
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5. Conclusions We found no increase in newly-formed, aging-related, neuroprotective
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WFA-positive PNNs surrounding PV neurons in the association cortex, which may be partially responsible for the increased susceptibility of the region to oxidative stress and dementia-linked damages. Furthermore, PV protein
in the entire cortex with aging.
AUTHOR CONTRIBUTIONS
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expression, which is essential for the generation of -oscillation, was decreased
Study concept and design: H.U., M.O., and T.I. Acquisition of data: H.U., K.T., and S.S. Analysis and interpretation of data: H.U., K.T., and S.S. Drafting of the
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manuscript: H.U. and M.O. Critical revision of the manuscript for important
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intellectual content: S.M., N.K., K.W., S.A., and T.I. Statistical analysis: H.U. and
Funding
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S.S. Study supervision: M.O., S.A., and T.I.
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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of interests The authors declare they have no competing financial interests.
Acknowledgements We thank Kawasaki Medical School Central Research Institute for making 19
ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
instruments available to support this work. We thank Y. Koshidaka for technical assistance. This work was supported in part by the Ryobi Teien Memory
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(www.editage000.jp) for English language editing.
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Foundation (H. Ueno). The authors would also like to thank Editage
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Zlokovic, B.V., 2011. Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders. Nat. Rev. Neurosci. 12, 723-738.
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FIGURE LEGENDS Fig. 1. Distribution of PV neurons and WFA-positive PNNs in the cerebral cortex
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in 2-month-old and 12-month-old mice Representative whole-brain sections labeled for parvalbumin (A) and WFA (B).
Parvalbumin in 2 months old (A, left panels), parvalbumin in 12 months old (A, right panels), WFA in 2 months old (B, left panels), and WFA in 12 months old
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mice (B, right panels) are shown. PV, parvalbumin-expressing interneurons; PNN, perineuronal nets; WFA, Wisteria floribunda agglutinin; FrA, frontal association cortex; DLO, dorsolateral orbital cortex; LO, lateral orbital cortex; VO, ventral orbital cortex; Cg, cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; DP, dorsal peduncular cortex; M1, primary motor cortex; M2, secondary motor cortex; MPtA, medial parietal association cortex; LPtA, lateral parietal
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association cortex; PTPR, parietal cortex, post, rostral; S1Tr, primary
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somatosensory cortex–trunk region; S1BF, primary somatosensory cortex– barrel field; S2, secondary somatosensory cortex; V2MM, secondary visual
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cortex–mediomedial area; V2ML, secondary visual cortex, mediolateral area; V1M, primary visual cortex, monocular area; V1B, primary visual cortex,
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binocular area; V2L, secondary visual cortex, lateral area; Au1, primary auditory cortex; AuD, secondary auditory cortex, dorsal area; AuV, secondary auditory cortex, ventral area; TeA, temporal association cortex; Ect, ectorhinal cortex; PRh,
perirhinal
cortex;
DIEnt,
dorsintermed
entorhinal
cortex;
DLEnt,
dorsolateral entorhinal cortex; RSD, retrosplenial dysgranular cortex; RSGc, retrosplenial granular cortex, c region; RSGb, retrosplenial granular cortex, b region; RSGa, retrosplenial granular cortex, a region. Scale bar = 1 mm.
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Fig. 2. The effect of aging on PV neurons and WFA-positive PNNs in the mouse
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cerebral cortex Representative double immunofluorescent images show the laminar distribution
of PV neurons (left panels) and WFA-positive PNNs (right panels) in the Au1 (A),
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S1BF (B), V1M (C), S2 (D), RSGc (E), MPtA (F), PL (G), Ect (H), and DLEnt (I)
at the 2-month-old (A–I, left panels) and 12-month-old stages (A–I, right panels). The same capture conditions were used for all section images. Every image shows a coronal section cut through the cerebral cortex with the pia at the top. White arrowheads indicate WFA-positive PNNs that do not contain PV. Black arrowheads indicate PV neurons without WFA-positive PNNs. Abbreviations are
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the same as in Figure 1. Scale bar = 100 µm in I (applies to A–I).
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Fig. 3. The effect of aging on the densities of PV neurons and WFA-positive PNNs in the mouse cerebral cortex
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Region-specific aging patterns of PV neuron density (A) and WFA-positive PNN density (B) in individual cortices are shown. Data are expressed as the mean ±
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SEM (n = 5 mice per group). *P < 0.05 for comparison between age groups in the same region. Abbreviations are the same as in Figure 1. Graphs show mean ± SEM (n = 5 mice per group).
Fig. 4. The effect of aging on the co-localization of PV and WFA in the mouse cerebral cortex Region-specific aging pattern of the percentage of PV neurons enveloped by
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WFA-positive PNNs (A) and the percentage of WFA-positive PNNs that contain PV (B) in individual cortices are shown. Data are expressed as the mean ± SEM
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(n = 5 mice per group). *P < 0.05 for comparison between age groups in the same region. Abbreviations are the same as in Figure 1. Graphs show mean ±
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SEM (n = 5 mice per group).
Fig. 5. The effect of aging on the mean fluorescence intensity of PV neurons in the mouse cerebral cortex
Region-specific aging patterns of the mean fluorescence intensity of PV neurons in individual cortices are shown. Data are expressed as the mean ± SEM (n = 5 mice per group). *P < 0.05 for comparison between age groups in the same region. Abbreviations are the same as in Figure 1. Graphs show mean ± SEM (n
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= 5 mice per group).
Fig. 6. The effect of aging on the soma area of PV neurons in the mouse
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cerebral cortex
Region-specific aging patterns of the soma area of PV neurons in individual
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cortices are shown. Data are expressed as the mean ± SEM (n = 5 mice per group). *P < 0.05 for comparison between age groups in the same region. Abbreviations are the same as in Figure 1. Graphs show mean ± SEM (n = 5 mice per group).
Fig. 7. The effect of aging on the co-localization of WFA-positive PNNs and GABAergic interneurons in the mouse primary somatosensory cortex
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Double confocal images for WFA (A–F, left panels), calbindin (A), calretinin (B), somatostatin (C), vasoactive-intestinal peptide (D), GAD67 (E), and NeuN (F) in
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the mouse primary somatosensory cortex at the 2-month-old (A–F, left panels) and 12-month-old stages (A–F, right panels) are shown. Arrowheads indicate
WFA-positive PNNs (E, F). Scale bar = 100 µm in F (applies to A–F). PNN,
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perineuronal nets; WFA, Wisteria floribunda agglutinin
Fig. 8. The effect of aging on the co-localization of WFA-positive PNNs and GABAergic interneurons in the mouse primary auditory cortex
Double confocal images for WFA (A–F, left panels), calbindin (A), calretinin (B), somatostatin (C), vasoactive-intestinal peptide (D), GAD67 (E), and NeuN (F) in the mouse primary auditory cortex at the 2-month-old (A–F, left panels) and
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12-month-old stages (A–F, right panels) are shown. Arrowheads indicate
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WFA-positive PNNs (E, F). Scale bar = 100 µm in F (applies to A–F).
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PNN, perineuronal nets; WFA, Wisteria floribunda agglutinin
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Highlights
・PV neuron density remained unchanged with aging in numerous cortical areas.
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・WFA-positive PNN density increased with aging in the sensory cortex.
・The expression level of PV protein in PV neurons decreased with aging.
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・The PV soma area was reduced with aging in numerous cortical areas.
S0197-0186(17)30349-2
DOI:
10.1016/j.neuint.2017.11.001
Reference:
NCI 4160
To appear in:
Neurochemistry International
Received Date: 14 June 2017 Revised Date:
28 October 2017
Accepted Date: 1 November 2017
Please cite this article as: Ueno, H., Takao, K., Suemitsu, S., Murakami, S., Kitamura, N., Wani, K., Okamoto, M., Aoki, S., Ishihara, T., Age-dependent and region-specific alteration of parvalbumin neurons and perineuronal nets in the mouse cerebral cortex, Neurochemistry International (2017), doi: 10.1016/j.neuint.2017.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
Research Article
Age-Dependent and Region-Specific Alteration of Parvalbumin
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Neurons and Perineuronal Nets in the Mouse Cerebral Cortex1
Hiroshi Uenoa, b, *, Keizo Takaoc, Shunsuke Suemitsud, Shinji Murakamid,
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Naoya Kitamurad, Kenta Wanid, Motoi Okamotob, Shozo Aokid, Takeshi Ishiharad
a
1
Department of Medical Technology, Kawasaki University of Medical Welfare,
Abbreviations: FrA, frontal association cortex; DLO, dorsolateral orbital cortex; LO, lateral orbital cortex;
VO, ventral orbital cortex; Cg, cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; DP, dorsal
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peduncular cortex; M1, primary motor cortex; M2, secondary motor cortex; MPtA, medial parietal
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association cortex; LPtA, lateral parietal association cortex; PTPR, parietal cortex, post, rostral; S1Tr, primary somatosensory cortex–trunk region; S1BF, primary somatosensory cortex–barrel field; S2,
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secondary somatosensory cortex; V2MM, secondary visual cortex–mediomedial area; V2ML, secondary visual cortex, mediolateral area; V1M, primary visual cortex, monocular area; V1B, primary visual cortex,
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binocular area; V2L, secondary visual cortex, lateral area; Au1, primary auditory cortex; AuD, secondary auditory cortex, dorsal area; AuV, secondary auditory cortex, ventral area; TeA, temporal association cortex; Ect, ectorhinal cortex; PRh, perirhinal cortex; DIEnt, dorsintermed entorhinal cortex; DLEnt, dorsolateral entorhinal cortex; RSD, retrosplenial dysgranular cortex; RSGc, retrosplenial granular cortex, c region; RSGb, retrosplenial granular cortex, b region; RSGa, retrosplenial granular cortex, a region; AD, Alzheimer’s Disease.
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ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
Okayama, 701-0193, Japan b
Department of Medical Technology, Graduate School of Health Sciences,
c
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Okayama University, Okayama, 700-8558, Japan Life Science Research Center, University of Toyama, Toyama, 930-0194, Japan
Department of Psychiatry, Kawasaki Medical School, Kurashiki, 701-0192,
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d
Japan
Author Information
Hiroshi Ueno, E-mail: [email protected] Keizo Takao, E-mail: [email protected]
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Shunsuke Suemitsu, E-mail: [email protected]
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Shinji Murakami, E-mail: [email protected] Naoya Kitamura, E-mail: [email protected]
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Kenta Wani, E-mail: [email protected] Motoi Okamoto, E-mail: [email protected]
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Shozo Aoki, E-mail: [email protected] Takeshi Ishihara, E-mail: [email protected]
*Corresponding author. Hiroshi Ueno, PhD. Address: Department of Medical Technology, Kawasaki University of Medical
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Welfare, 288, Matsushima, Kurashiki, Okayama, 701-0193, Japan Phone: +81-86-462-1111, Fax: +81-86-462-1193
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E-mail address: [email protected] (H. Ueno)
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ABSTRACT Cognitive function declines with age. Such function depends on in
the
frontal
cortex.
Pyramidal
neurons,
and
the
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-oscillation
parvalbumin-expressing interneurons (PV neurons) that control them, are important for the generation of -oscillation. The mechanism by which
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cognitive function declines is unclear. Perineuronal nets (PNNs) mainly surround the soma and proximal dendrites and axon segments of PV neurons in the cerebral cortex. Previous evidence indicates that PNNs inhibit neural plasticity. If this is true, an increase in the number of neurons surrounded by PNNs or in the thickness or density of the PNNs around neurons could decrease plasticity in the cortex. To determine if an aging-related change in cortical PNNs occurs, we examined the influence aging
on
PV
neurons
and
whether
Wisteria
floribunda
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of
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agglutinin-positive PNNs differ depending on the cortical area. The results showed that the number of PV neurons/mm2 did not change in many areas
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of the cortex as mice aged. In contrast, the number of neurons in the sensory cortex surrounded by PNNs increased as mice aged. Thus, with
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age, PNN density increases in some cortical areas but not in others. In addition, the expression level of PV protein in PV neurons decreased with aging in the whole cortex. We suggest that decreased expression of PV protein impairs fast spiking in PV neurons. We propose that PNNs surround more neurons as age increases. This aging-related increase in PNNs decreases plasticity in the cerebral cortex and reduces cognitive function. The first step in investigating this proposal would be to
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determine if PNN density increases with age.
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Keywords: aging, cerebral cortex, mouse, parvalbumin, perineuronal nets
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1. Introduction As animals naturally age, their cognitive function declines; however, the
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underlying mechanisms driving this process have not been elucidated. No one has clarified yet whether aging-related change occurs in the whole brain or in
localized brain regions. Cognitive function in the brain depends on γ-oscillation, in
turn
depends on
the
function
of
pyramidal neurons and
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which
parvalbumin-expressing interneurons (PV neurons (Sohal et al., 2009; Kleschevnikov et al., 2012; Lewis et al., 2005).
PV neurons are one category of GABAergic interneurons among several categories. We can distinguish different types of GABAergic neurons by noting which calcium-binding protein (CaBP) they express: parvalbumin, calbindin, or calretinin (Markram et al., 2004). Categorizing interneurons according to their
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calcium-binding protein is useful because these neurons exhibit a wide variety of
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other attributes such as morphology, physiology, and the expression of other molecules. The type of GABAergic interneuron that expresses parvalbumin
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accounts for about half of all GABAergic interneurons in the cerebral cortex (Rudy et al., 2011). A single PV neuron makes synapses with hundreds of
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pyramidal neurons and controls the synchronous firing of these neurons (Kawaguchi, 2001). This is essential for the generation of -oscillations related to working memory (Bartos et al., 2007; Sohal et al., 2009). Therefore, a decrease in PV protein level leads to an impairment in the frequent firing of PV neurons and a loss of -oscillatory activity (Sohal et al., 2009; Volman et al., 2011). Previous work suggests that PV neurons cause functional deterioration with age and accelerate cognitive impairment (Lewis, 2012; Takahasi, 2002).
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In the cortex, approximately 80% of PV neurons are surrounded by extracellular matrix molecules (Berretta et al., 2015). These molecules form a
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mesh-like structure, called a perineuronal net (PNN). PNNs surround the cell bodies, proximal dendrites, and initial axon segments of PV neurons. They are mainly composed of chondroitin sulfate proteoglycans (CSPGs), hyaluronic acid,
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and link proteins (Bandtlow and Zimmermann, 2000; Dityatev and Schachner, 2003). CSPGs expressed in the central nervous system include lectican, aggrecan, brevican, and neurocan. PNNs
mainly
function
in
controlling
synaptic
plasticity
and
in
neuroprotection (Wang and Fawcett, 2012). They appear during development, near the end of the critical period, in the sensory cortex (Chevaleyre and Piskorowski, 2014; Sun, 2009). Dissolving PNNs with chondroitinase ABC in the
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primary visual cortex of adult rats induced experience-driven plasticity, similar to
2002).
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the plasticity observed in young rats during the critical period (Pizzorusso et al.,
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Many studies have indicated that the brain exhibits a decrease in plasticity with age (Gray and Barnes, 2015; Mora, 2013; Lehmann et al., 2012). If PNNs
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do indeed inhibit plasticity, then an aging-related increase in the number of neurons surrounded by PNN would reduce plasticity, and maybe cognitive function, in older individuals. In this study, we looked for age-related increases in the number of PNNs in the cerebral cortex to determine if such changes are correlated with cognitive decline.
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2. Materials and methods 2.1. Animals
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We used male mice (C57BL/6) for the experiments. The day of birth was designated as postnatal day 0. We divided the mice into three groups according
to age: 2 months postnatally (2 months, n =5), 6 months postnatally (6 months, n
= 5), and 12 months postnatally (12 months, n = 5). We purchased the animals
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from Charles River Laboratories (Kanagawa, Japan) and housed them in cages (3–5 animals per cage) with food and water provided ad libitum under a 12-h light/dark cycle at 23°C–26°C. We made every effort to minimize the number of animals used and their suffering. These experiments complied with the U.S. National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised in 1996) and were approved by the
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Committee for Animal Experiments at Kawasaki Medical School Advanced
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Research Center.
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2.2. Tissue preparation
For tissue preparation from mice (2, 6, and 12 months old), we deeply
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anesthetized the animals with a lethal dose of sodium pentobarbital (120 mg/kg, i.p.) and transcardially perfused them with ice-cold phosphate-buffered saline (PBS) for 2 min and then 4% paraformaldehyde in PBS (pH 7.4) for 10 min (10 ml/min). In all cases, we dissected all the brains and post-fixed them overnight with 4% paraformaldehyde in PBS at 4°C and cryoprotected them by immersion in 15% sucrose for 12 h followed by 30% sucrose for 20 h at 4°C. To cut sections, we froze the brains in O.C.T. Compound (Tissue-Tek; Sakuma Finetek, Tokyo,
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Japan) using dry ice-cold normal hexane and we prepared serial coronal sections of 40-µm thickness using a cryostat (CM3050S; Leica Wetzlar,
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Germany) at -20°C. We collected sections in ice-cold PBS containing 0.05% sodium azide.
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2.3. Immunohistochemistry
We visualized PNNs with antibodies that recognize sugar chains. The lectins Wisteria floribunda agglutinin (WFA) and Vicia villosa agglutinin bind to the terminal N-acetylgalactosamine residues of CSPGs (Young and Williams, 1985). Lectin WFA has been frequently used as a comprehensive marker of PNNs. We treated the cryostat sections with 0.1% Triton X-100 in PBS at room temperature for 15 min. After three washes in PBS, we incubated the sections with 10%
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normal goat serum (ImmunoBioScience Corp., Mukilteo, WA) in PBS at room
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temperature for 1 h, we washed them three times in PBS, and incubated them overnight at 4°C in PBS containing biotinylated WFA (B-1355, Vector
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Laboratories, Funakoshi Co., Tokyo, Japan; 1:200) and primary antibodies (described below). After washing in PBS, we incubated the sections with secondary
antibodies
(indicated
below)
and
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corresponding
streptavidin-conjugated Texas Red (SA-5006; Vector Laboratories) at room temperature for 2 h. We rinsed the labeled sections again with PBS and we mounted them on glass slides with Vectashield medium (H-1400; Vector Laboratories). We stored the prepared slides at 4°C until we used them in the microscopy analysis.
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ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
2.4. Antibodies We used the following primary antibodies for staining: mouse anti-parvalbumin
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(clone PARV-19, P3088; Sigma-Aldrich Japan, Tokyo, Japan; 1:1,000), rabbit anti-calbindin (ab25085; Abcam, Tokyo, Japan; 1:400), mouse anti-calretinin (clone
6B8.2,
MAB1568;
Millipore,
Tokyo,
Japan;
1:500),
rabbit
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anti-somatostatin (T-4103; Peninsula Laboratories, Belmont, CA; 1:2,000),
rabbit anti-vasoactive-intestinal peptide (20077; Immunostar, Hudson, WI; 1:1,000), mouse anti-GAD67 (clone 1G10.2, MAB5406; Millipore; 1:1,000), and mouse anti-NeuN (clone A60, MAB377; Millipore; 1:500). We used the following secondary antibodies for visualization: Alexa Fluor 488-conjugated goat anti-mouse IgG (ab150113; Abcam; 1:1,000) and Alexa Fluor 488-conjugated
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2.5. Microscopy imaging
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goat anti-rabbit IgG (ab150077; Abcam; 1:1,000).
For quantification of the number and fluorescence intensity of PV- and
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WFA-positive neurons, we used confocal laser scanning microscopy to obtain images of stained sections. Images (1024 × 1024 pixels) were saved as TIFF
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files using ZEN software. Briefly, we performed the analysis was performed using a × 10 or × 20 objective lens and a pinhole setting that corresponded to a focal plane thickness of less than 1 m. Images from whole sections using a × 10 objective lens of the fluorescence microscope (BZ-X; KEYENCE, Tokyo, Japan) and we merged them using KEYENCE BZ-X Analyzer software (KEYENCE).
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2.6. Quantification of labeled neurons In this experiment, we measured three attributes of labeled neurons in the
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cortices of the mice. 1) The density of labeled neurons. For this, we counted the number of labeled neurons in a cortical region of the cortex (defined by the mouse atlas; Paxinos and Franklin, 2012) and we measured the area of that
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region. We then calculated labeled neurons/mm 2. 2) The distribution of labeled
neurons. For this, we examined the distribution across the different cortical regions that we studied. 3) The fluorescence intensity of labeled neurons. For this, we selected 12 sections from each mouse brain and stained them as described above. On 8-bit images of each section, we manually outlined each PV-positive neuron cell body and measured the gray level with NIH ImageJ (NIH, Bethesda, MD; http://rsb.info.nih.gov/nih-image/). Prior to capture, the exposure
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time, gain, and offset were carefully set to ensure a strong signal but to avoid
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saturation. Identical capture conditions were used for all sections. Background intensity was subtracted using unstained portions of each section. We acquired
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all confocal images as TIFF files and analyzed them with NIH ImageJ. We coded the slides and a blinded independent observer quantified them. We performed
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estimations of neuronal density (cells/mm2).
2.7. Data analysis
Data are expressed as mean ± S.E.M. Statistical significance was determined using one-way ANOVA with Bonferroni multiple comparison test, and was set at *p < 0.05.
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3. Results 3.1. Effect of aging on PV neurons and WFA-positive PNNs in the mouse
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cerebral cortex As shown in Figures 1A and 2 (green) the fluorescence intensity of PV neurons was lower in 12-month-old mice. As shown in Figures 1B and 2 (red),
the laminar distribution of WFA-positive PNNs throughout the cortical layers was
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very similar in 2-month- and 12-month-old mice. Weaker PV immunoreactivity was observed in all 12-month-old mouse cortices examined, compared to 2-month-old mouse cortices.
3.2. Effect of aging on PV neuron density and WFA-positive PNN density in
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the mouse cerebral cortex
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Figure 3A shows the density of PV neurons in each of the cortical areas that we examined and for each age. In most of the 33 areas that we examined, there
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was no significant difference between the density of PV neurons at different ages. In three areas, Cg/M2. AuD, and RSGb, the density of PV neurons was
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significantly higher in 12-month-old mice than in 2-month-old mice. Figure 3B shows the density of WFA-positive PNNs in each of the cortical
areas that we examined and for each age. In some of the areas that we examined, there was no significant difference between the density of WFA-positive PNNs at different ages. In other areas, however, the density in 12-month-old mice was significantly higher than that in 2-month-old mice. These regions were: frontal areas: VO, Cg, M2; parietal areas: MPtA, S1BF; occipital
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areas: V2MM, V1B; auditory temporal cortex: Au1, AuD, AuV; other temporal
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cortex: TeA, DIEnt; retrosplenial cortex: RSGb.
3.3. Effect of aging on the co-localization of PV neurons and WFA-positive PNNs in the mouse cerebral cortex
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As Figure 4A shows, there was no significant difference in this percentage
between 2-month- and 12-month-old mice in most of the areas that we examined. The percentage was significantly higher in 12-month-old mice in the following four areas: frontal - Cg; parietal - S1Tr; occipital - V2MM; auditory temporal AuD.
Figure 4B shows the percentage of WFA-positive PNNs that covered PV neurons in each of the cortical areas that we examined and for each age. In
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some of the areas that we examined, there was no significant difference
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between the percentage of WFA-positive PNNs that covered PV neurons at different ages. In other areas, however, the percentage in 12-month-old mice
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was significantly lower than that in 2-month-old mice. These regions were: parietal areas: S1BF, S2; occipital areas: V2MM, V1B, V2L; auditory temporal
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cortex: Au1, AuD; retrosplenial cortex: RSD, RSGb. Newly formed WFA-positive PNNs did not cover PV neurons in these areas in 12-month-old mice.
3.4. Effect of aging on the fluorescence intensity of PV neurons in the mouse cerebral cortex To examine the effect of aging on the expression of PV protein in the brain, we analyzed the fluorescence intensity of each PV-positive neuronal soma in
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ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
each of the mouse cortical areas we examined. As shown in Figure 5, the fluorescence intensity of PV neurons was significantly lower in 12-month-old
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mice than in 2-month-old mice. Figure 5 also shows that the intensity of PV labeling differed in each of the cortical areas that we examined and for each age.
These results raise the possibility that the possibility that PV neuron function
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differs significantly between 2 months and 12 months of age in mice.
3.5. Effect of aging on the soma area of PV neurons in the mouse cerebral cortex
The area of the soma in PV-labeled neurons did not significantly differ between 2-month-old and 12-month-old mice in 23 of the 33 brain areas that we examined. In 10 brain areas, the size of each PV-positive soma was smaller in
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12-month-old mice than in 2-month-old mice. These 10 areas were: frontal: Vo,
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M2; parietal: S2; occipital: V2MM, V1M, V2L; auditory temporal: AuD; other
3.6.
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temporal: DLEnt; retrosplenial: RSGa, RSGb.
Co-localization
of
newly
formed
WFA-positive
PNNs
and
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GAD67-expressing neurons in the mouse primary somatosensory cortex and primary auditory cortex with aging In both the primary somatosensory cortex (S1BF) and primary auditory
cortex (Au1) of 12-month-old mice, WFA-positive PNNs formed around neurons that were not PV neurons (Fig. 2A–B, 4). To further characterize the neurons surrounded by WFA-positive PNNs in areas S1BF and Au1 of 12-month-old mice, we stained for calbindin (CB), calretinin (CR), somatostatin (SOM), and
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ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
vasoactive-intestinal peptide (VIP) (Fig. 7A–D, 8A–D). In both the primary somatosensory cortex and primary auditory cortex, CB, CR, SOM, and VIP
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neurons were not surrounded by WFA-positive PNNs from the 6-month to the 12-month stages (Fig. 7A–D, 8A–D). Next, we labeled GABAergic neurons
using the anti-GAD67 antibody (Kaufman et al., 1991). In both the primary
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somatosensory cortex and primary auditory cortex of 12-month-old mice, we
observed that some of the WFA-positive PNNs did not contain GAD67 (Fig. 7E, 8E). In addition, we examined which cells surrounded by WFA-positive PNNs that are formed in the adult brain co-localized with NeuN. NeuN was used to identify neurons to exclude non-neuronal cells. Most cells enveloped by WFA-positive PNNs in the 12-month-old primary sensory cortex did not contain NeuN (Fig. 7F, 8F). In some brain regions, some groups of neurons do not
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express NeuN (Mullen et al., 1992, Weyer and Schilling, 2003). In the
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cerebellum, these neurons coincide with GABAergic interneurons (Weyer and Schilling, 2003). According to the distribution and shape of the neurons, the
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findings suggested that the neurons formed by WFA-positive PNNs in the adult primary sensory cortices are GABAergic interneurons, which do not contain
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GAD67.
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4. Discussion This study showed that the influence of aging on PV neurons and
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WFA-positive PNNs differs depending on the cortical area. The number of PNN-positive neurons increased more with age in the primary sensory cortices
than in other cortical areas. Aging-related increases in the percentage of PV
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neurons that are surrounded by PNNs in the association cortices, such as in the
frontal and entorhinal cortices, were not observed. The PV neuron density also did not change in numerous cortical areas. The expression of PV protein was significantly reduced in the aged cortices examined in this study compared to the cortices of 2-month-old mice.
Cognitive impairment with aging is caused by decreases in the numbers of neurons and dendrites in a smaller and more limited area (Burke and Barnes,
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2006). In line with previous findings that cognitive function and memory decline
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with age although neuronal numbers are preserved (Rapp et al., 2002; Freeman et al., 2008; Burke and Barnes, 2006) and that PV neuron density did not
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decrease with aging in the sensory cortices (Karetko-Sysa et al., 2014), we found that PV neuron density remained unchanged with aging in numerous
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cortical areas.
Intracellular Ca2+ greatly influences the physiological, chemical, and
biological processes associated with neurons (Foster, 2007). Many studies have shown that Ca2+ conductance increases with age (Farajnia et al., 2015). It has also been reported that Ca2+ homeostasis is disrupted in the prefrontal cortex of aged monkeys (Chang et al., 2005). PV, a Ca2+-binding protein, is important for regulating intracellular Ca2+ kinetics in PV-expressing neurons (Chard et al,
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ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
1993; Baimbridge et al., 1992). Moreover, a previous study indicated that Ca2+ signaling in PV neurons decreased with age in mice (Jessen et al., 2017), but
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there has been no report of decreased PV protein expression in aged mice. Reduction of PV expression reduces the high-frequency firing of PV neurons
and leads to the loss of -oscillation by PV neurons (Lewis, 2012; Murray et al.,
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2015).
Decreases in sensory input are partially responsible for aging-related decline in PV expression, as PV protein expression in sensory cortical PV neurons depends on sensory input (Patz et al., 2004) and mice show less movement and social behavior with aging (Shoji et al., 2016). Another possible reason may be the aging-dependent decline in brain-derived neurotrophic factor (BDNF) expression in the central nervous system (Adlard et al., 2005; Gooney et
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al., 2004), as BDNF is essential for the expression of PV mRNA in PV neurons
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during development (Huang et al., 1999; Zheng et al., 2011). In line with the results of a previous study (Peinado et al., 1997), we showed
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that the soma areas of PV neurons shrink in numerous cortical areas with aging. In other words, the aging-dependent soma shrinking in PV neurons may reflect
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functional deterioration.
For the first time, we showed that the aging-related increase in
WFA-positive PNN density is region-selective, occurring in the somatosensory, visual, and auditory cortices, rather than in the association cortex. We did not observe aging-related increase in WFA-positive PNN density in areas requiring high flexibility throughout the lifespan, such as the frontal cortex, and we suggest that the plasticity of the association cortex is preserved with aging.
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It remains unclear which types of neurons WFA-positive PNNs surround with aging. In this study, it was shown that these neurons are not positive for CB,
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CR, SOM, VIP, GAD67, and NeuN; a previous study suggested that they may be GAD67-negative GABAergic interneurons (Karetko-Sysa et al., 2014). Based on their morphology,
size,
and
distribution,
the
neurons
surrounded
by
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newly-formed, aging-related, WFA-positive PNNs may be PV-negative PV
neurons or PV neurons expressing PV at levels lower than the detection threshold. Further studies are needed to determine which neurons are surrounded by newly-formed, aging-related, WFA-positive PNNs.
PNN function in protecting neurons from oxidative stress, tau protein toxicity, and glutamic acid excitotoxicity is well documented (Okamoto et al., 1994; Morawski et al., 2010; Morawski et al., 2014; Suttkus et al., 2014 ). We found
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that there was no aging-related increase in PV neurons surrounded by PNNs in
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the association cortex. Furthermore, PV protein expression, which prevents excessive Ca2+ intracellular influx, was found to decrease with aging. A recent
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study found that -oscillation involving PV neurons in the entorhinal cortex was reduced at the early stage of Alzheimer’s disease (AD) (Klein et al., 2016).
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Moreover, reactive oxygen species increase in the brain with aging (Balbi et al., 2015; Zlokovic, 2011), which may promote dementia (de la Torre and Stefano, 2000). The present results indicate that the association cortex, which contains numerous PV neurons without neuroprotective PNNs, may be particularly susceptible to oxidative stress and dementia-related damages with aging; suggesting that there is region-selective vulnerability in the cortex.
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5. Conclusions We found no increase in newly-formed, aging-related, neuroprotective
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WFA-positive PNNs surrounding PV neurons in the association cortex, which may be partially responsible for the increased susceptibility of the region to oxidative stress and dementia-linked damages. Furthermore, PV protein
in the entire cortex with aging.
AUTHOR CONTRIBUTIONS
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expression, which is essential for the generation of -oscillation, was decreased
Study concept and design: H.U., M.O., and T.I. Acquisition of data: H.U., K.T., and S.S. Analysis and interpretation of data: H.U., K.T., and S.S. Drafting of the
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manuscript: H.U. and M.O. Critical revision of the manuscript for important
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intellectual content: S.M., N.K., K.W., S.A., and T.I. Statistical analysis: H.U. and
Funding
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S.S. Study supervision: M.O., S.A., and T.I.
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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of interests The authors declare they have no competing financial interests.
Acknowledgements We thank Kawasaki Medical School Central Research Institute for making 19
ACCEPTED MANUSCRIPT Hiroshi Ueno et al.
instruments available to support this work. We thank Y. Koshidaka for technical assistance. This work was supported in part by the Ryobi Teien Memory
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(www.editage000.jp) for English language editing.
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Foundation (H. Ueno). The authors would also like to thank Editage
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FIGURE LEGENDS Fig. 1. Distribution of PV neurons and WFA-positive PNNs in the cerebral cortex
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in 2-month-old and 12-month-old mice Representative whole-brain sections labeled for parvalbumin (A) and WFA (B).
Parvalbumin in 2 months old (A, left panels), parvalbumin in 12 months old (A, right panels), WFA in 2 months old (B, left panels), and WFA in 12 months old
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mice (B, right panels) are shown. PV, parvalbumin-expressing interneurons; PNN, perineuronal nets; WFA, Wisteria floribunda agglutinin; FrA, frontal association cortex; DLO, dorsolateral orbital cortex; LO, lateral orbital cortex; VO, ventral orbital cortex; Cg, cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; DP, dorsal peduncular cortex; M1, primary motor cortex; M2, secondary motor cortex; MPtA, medial parietal association cortex; LPtA, lateral parietal
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association cortex; PTPR, parietal cortex, post, rostral; S1Tr, primary
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somatosensory cortex–trunk region; S1BF, primary somatosensory cortex– barrel field; S2, secondary somatosensory cortex; V2MM, secondary visual
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cortex–mediomedial area; V2ML, secondary visual cortex, mediolateral area; V1M, primary visual cortex, monocular area; V1B, primary visual cortex,
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binocular area; V2L, secondary visual cortex, lateral area; Au1, primary auditory cortex; AuD, secondary auditory cortex, dorsal area; AuV, secondary auditory cortex, ventral area; TeA, temporal association cortex; Ect, ectorhinal cortex; PRh,
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cortex;
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cortex;
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dorsolateral entorhinal cortex; RSD, retrosplenial dysgranular cortex; RSGc, retrosplenial granular cortex, c region; RSGb, retrosplenial granular cortex, b region; RSGa, retrosplenial granular cortex, a region. Scale bar = 1 mm.
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Fig. 2. The effect of aging on PV neurons and WFA-positive PNNs in the mouse
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cerebral cortex Representative double immunofluorescent images show the laminar distribution
of PV neurons (left panels) and WFA-positive PNNs (right panels) in the Au1 (A),
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S1BF (B), V1M (C), S2 (D), RSGc (E), MPtA (F), PL (G), Ect (H), and DLEnt (I)
at the 2-month-old (A–I, left panels) and 12-month-old stages (A–I, right panels). The same capture conditions were used for all section images. Every image shows a coronal section cut through the cerebral cortex with the pia at the top. White arrowheads indicate WFA-positive PNNs that do not contain PV. Black arrowheads indicate PV neurons without WFA-positive PNNs. Abbreviations are
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the same as in Figure 1. Scale bar = 100 µm in I (applies to A–I).
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Fig. 3. The effect of aging on the densities of PV neurons and WFA-positive PNNs in the mouse cerebral cortex
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Region-specific aging patterns of PV neuron density (A) and WFA-positive PNN density (B) in individual cortices are shown. Data are expressed as the mean ±
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SEM (n = 5 mice per group). *P < 0.05 for comparison between age groups in the same region. Abbreviations are the same as in Figure 1. Graphs show mean ± SEM (n = 5 mice per group).
Fig. 4. The effect of aging on the co-localization of PV and WFA in the mouse cerebral cortex Region-specific aging pattern of the percentage of PV neurons enveloped by
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WFA-positive PNNs (A) and the percentage of WFA-positive PNNs that contain PV (B) in individual cortices are shown. Data are expressed as the mean ± SEM
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(n = 5 mice per group). *P < 0.05 for comparison between age groups in the same region. Abbreviations are the same as in Figure 1. Graphs show mean ±
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SEM (n = 5 mice per group).
Fig. 5. The effect of aging on the mean fluorescence intensity of PV neurons in the mouse cerebral cortex
Region-specific aging patterns of the mean fluorescence intensity of PV neurons in individual cortices are shown. Data are expressed as the mean ± SEM (n = 5 mice per group). *P < 0.05 for comparison between age groups in the same region. Abbreviations are the same as in Figure 1. Graphs show mean ± SEM (n
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= 5 mice per group).
Fig. 6. The effect of aging on the soma area of PV neurons in the mouse
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cerebral cortex
Region-specific aging patterns of the soma area of PV neurons in individual
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cortices are shown. Data are expressed as the mean ± SEM (n = 5 mice per group). *P < 0.05 for comparison between age groups in the same region. Abbreviations are the same as in Figure 1. Graphs show mean ± SEM (n = 5 mice per group).
Fig. 7. The effect of aging on the co-localization of WFA-positive PNNs and GABAergic interneurons in the mouse primary somatosensory cortex
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Double confocal images for WFA (A–F, left panels), calbindin (A), calretinin (B), somatostatin (C), vasoactive-intestinal peptide (D), GAD67 (E), and NeuN (F) in
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the mouse primary somatosensory cortex at the 2-month-old (A–F, left panels) and 12-month-old stages (A–F, right panels) are shown. Arrowheads indicate
WFA-positive PNNs (E, F). Scale bar = 100 µm in F (applies to A–F). PNN,
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perineuronal nets; WFA, Wisteria floribunda agglutinin
Fig. 8. The effect of aging on the co-localization of WFA-positive PNNs and GABAergic interneurons in the mouse primary auditory cortex
Double confocal images for WFA (A–F, left panels), calbindin (A), calretinin (B), somatostatin (C), vasoactive-intestinal peptide (D), GAD67 (E), and NeuN (F) in the mouse primary auditory cortex at the 2-month-old (A–F, left panels) and
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12-month-old stages (A–F, right panels) are shown. Arrowheads indicate
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WFA-positive PNNs (E, F). Scale bar = 100 µm in F (applies to A–F).
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PNN, perineuronal nets; WFA, Wisteria floribunda agglutinin
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Highlights
・PV neuron density remained unchanged with aging in numerous cortical areas.
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・WFA-positive PNN density increased with aging in the sensory cortex.
・The expression level of PV protein in PV neurons decreased with aging.
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・The PV soma area was reduced with aging in numerous cortical areas.