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Shati/Nat8l deficiency disrupts adult neurogenesis and causes attentional impairment through dopaminergic neuronal dysfunction in the dentate gyrus

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Abstract

Successful completion of daily activities relies on the ability to select the relevant features of the environment for memory and recall. Disruption to these processes can lead to various disorders, such as attention‐deficit hyperactivity disorder (ADHD). Dopamine is a neurotransmitter implicated in the regulation of several processes, including attention. In addition to the higher‐order brain function, dopamine is implicated in the regulation of adult neurogenesis. Previously, we generated mice lacking Shati, an N‐acetyltransferase‐8‐like protein on a C57BL/6J genetic background (Shati/Nat8l−/−). These mice showed a series of changes in the dopamine system and ADHD‐like behavioral phenotypes. Therefore, we hypothesized that deficiency of Shati/Nat8l would affect neurogenesis and attentional behavior in mice. We found aberrant morphology of neurons and impaired neurogenesis in the dentate gyrus (DG) of Shati/Nat8l−/− mice. Additionally, research has suggested that impaired neurogenesis might be due to the reduction of dopamine in the hippocampus. Galantamine (GAL) attenuated the attentional impairment observed in the object‐based attention test via increasing the dopamine release in the hippocampus of Shati/Nat8l−/− mice. The α7 nicotinic acetylcholine receptor (nAChR) antagonist, methyllycaconitine, and dopamine D1 receptor antagonist, SCH23390, blocked the ameliorating effect of GAL on attentional impairment in Shati/Nat8l−/− mice. These results suggest that the ameliorating effect of GAL on Shati/Nat8l−/− attentional impairment is associated with activation of D1 receptors following increased dopamine release in the hippocampus via α7 nAChR. In summary, Shati/Nat8l is important in both morphogenesis and neurogenesis in the DG and attention, possible via modulation of dopaminergic transmission.
Journal of Neurochemistry. 2020;00:1–14. wileyonlinelibrary.com/journal/jnc
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  1© 2020 International Society for Neurochemistry
Received: 10 March 2020 
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  Revised: 25 March 2020 
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  Accepted: 25 March 2020
DOI: 10.1111/jnc.1502 2
ORIGINAL ARTICLE
Shati/Nat8l deficiency disrupts adult neurogenesis and causes
attentional impairment through dopaminergic neuronal
dysfunction in the dentate gyrus
Bolati Wulaer1,2 | Kazuo Kunisawa3| Kazuhiro Hada4| Willy Jaya Suento2,5|
Hisayoshi Kubota3| Tsubasa Iida3| Aika Kosuge3| Taku Nagai4|
Kiyofumi Yamada4,7| Atsumi Nitta6,7| Yasuko Yamamoto2| Kuniaki Saito1,2,7 |
Akihiro Mouri3,7 | Toshitaka Nabeshima1,7
Bolati Wu laer and Kazuo Ku nisawa contri buted e qually t o this work
Abbreviations: ANOVA, anal ysis of variance ; BrdU, 5-bromo-2'- deoxy uridine; CA , cornu ammoni s; DCX , doubl ecort in; DG, dentat e gyrus; GAL , galantamin e; GFAP, glia l fibrillar y acidic
protein ; i.p., in trape ritoneally; M LA, methy llycac onitine; NA A, N-acetyla spart ate; NAc, nucl eus accumbens ; nAChR , nicotinic acet ylcholine re ceptor; NeuN , neuron s; OBAT,
objec t-base d attention tes t; PBS, phosp hate buf fered saline ; PCR, Re al-time quanti tative revers e trans cription poly merase chain r eaction; PFC , prefro ntal cortex; R AC, raclop ride; RRID,
Resear ch Resource Ide ntifiers (see s cicrunch.or g); SCH, SCH233 90; Shati/Nat8 l, N-acetyltransferase-8-like protein; Shati/Nat8l−/−, Shat i/Nat8l-knoc kout; Shati/Na t8l−/−, Sh ati/
Nat8l-k nockout; SOX 2, SRY-box-cont aining gene 2; TH , tyrosine hydr oxylase; WT, wil d type.
1Advanced Diagnostic Sys tem Research
Labor atory, Fujita Health Universit y
Gradu ate Scho ol of Health Science, Aichi,
Japan
2Depar tment of Disease Control and
Preventi on, Fujit a Health Univer sity
Gradu ate Scho ol of Health Science, Aichi,
Japan
3Depar tment of Regulato ry Science
for Evaluation & Deve lopment of
Pharma ceutic als & Devices, Fujita Health
University Graduate School of Health
Science , Aichi, Japan
4Department of Neuropsychopharmacology
and Hospital Pharmac y, Nagoya Universit y
Gradu ate Scho ol of Medicine, Ai chi, Jap an
5Depar tment of Psychiatry, Hasanuddin
University, Sout h Sulawesi, Indo nesia
6Depar tment of Pharma ceutic al Therapy
and Neuropharmacology, Graduate School
of Pharma ceutic al Scie nces, University of
Toyama, Toyama, Japan
7Japanese D rug Organization of Appropriate
Use and Res earch, Aichi, J apan
Correspondence
Akihiro Mouri, Depar tment of Regulatory
Science f or Evaluation and D evelopment
of Pharma ceutic als and D evices , Fujita
Health U niversity Gr aduate School of Health
Science s, Aichi, 470-1192, Japan.
Email: mouri@fujita-hu.ac.jp
Funding information
Japan Society for the Promotion of Science,
Abstract
Successful completion of daily activities relies on the ability to select the relevant
features of the environment for memory and recall. Disruption to these processes can
lead to various disorders, such as attention-deficit hyperactivity disorder (ADHD).
Dopamine is a neurotransmitter implicated in the regulation of several processes,
including attention. In addition to the higher-order brain function, dopamine is im-
plicated in the regulation of adult neurogenesis. Previously, we generated mice lack-
ing Shati, an N-acetyltransferase-8-like protein on a C57BL/6J genetic background
(Shati/Nat8l−/−). These mice showed a series of changes in the dopamine system and
ADHD-like behavioral phenotypes. Therefore, we hypothesized that deficiency of
Shati/Nat8l would affect neurogenesis and attentional behavior in mice. We found
aberrant morphology of neurons and impaired neurogenesis in the dentate gyrus of
Shati/Nat8l−/− mice. Additionally, research has suggested that impaired neurogenesis
might be because of the reduction of dopamine in the hippocampus. Galantamine
(GAL) attenuated the attentional impairment observed in the object-based attention
test via increasing the dopamine release in the hippocampus of Shati/Nat8l−/− mice.
The α7 nicotinic acetylcholine receptor antagonist, methyllycaconitine, and dopamine
D1 receptor antagonist, SCH23390, blocked the ameliorating effect of GAL on atten-
tional impairment in Shati/Nat8l−/− mice. These results suggest that the ameliorating
effect of GAL on Shati/Nat8l−/− attentional impairment is associated with activation
of D1 receptors following increased dopamine release in the hippocampus via α7 nic-
otinic acetylcholine receptor. In summary, Shati/Nat8l is important in both morpho-
genesis and neurogenesis in the dentate gyrus and attention, possible via modulation
of dopaminergic transmission.
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1 | INTRODUCTION
One of the most remarkable characteristics of the adult brain is its
ability to adapt to internal and external stimuli, which regulate in dy-
namic neuroplasticity phenomena. These neural plasticity processes
include changes in dendritic morphology and the generation of new
cells (Dioli et al., 2019). Pathological conditions are known to impair
brain plasticity by causing changes in dendritic length and arboriza-
tion, and also by decreasing the number of new neurons (Bangasser
et al., 2017; Dioli et al., 2017; Mateus-Pinheiro et al., 2013; Sousa
& Almeida, 2012). In adult mammals, new neurons are continu-
ously generated and are restricted primarily to the subventricular
zone and dentate gyrus (DG) in the hippocampus and the neuro-
genesis proceeds in the DG but not in the cornu ammonis regions
(Kempermann, 2006; Mahar, Bambico, Mechawar, & Nobrega, 2014;
Ming & Song, 2005). Adult hippocampal neurogenesis originates
from neuronal precursor cells in the DG and result s in new gran-
ule cell neurons after division, elimination, differentiation, and
maturation. The differential step of neurogenesis can be affected
by a variet y of factors, including the environment, pharmacological
interventions, exercise, and neurotransmitters, such as dopamine
(Baker, Baker, & Hagg, 2004; Borta & Hoglinger, 20 07; Hoglinger
et al., 2004; Zhang et al., 2018).
Dopamine is involved in higher-order brain functions, such as
motivation and attention. Several studies have suggested that do-
pamine controls differential steps of adult hippocampal neurogen-
esis (Baker et al., 2004; Borta & Hoglinger, 2007). In a lesion study,
depletion of dopamine in rodents decreased the proliferation and
survival of neural precursor cells in the DG; however, exposure to
dopamine antagonists has been reported to enhance proliferation
and neurogenesis (Hoglinger et al., 20 04; Niwa et al., 2007; Park &
Enikolopov, 2010). These conflicting results reflect a complex and
multifunctional nature of dopamine signaling in adult neurogenesis
(Park & Enikolopov, 2010).
In our previous studies, we identified a novel molecule, Shati,
an N-acetyltransferase-8-like protein (Shati/Nat8l) that synthesizes
N-acetylaspartate (NAA) from aspartate and acetyl-coenz yme A
(Ariyannur et al., 2010; Niwa et al., 2007). Shati inhibits metham-
phetamine-induced hyperlocomotion, sensitization, and conditioned
place preference and attenuates methamphetamine-induced dopa-
mine increases in the nucleus accumbens (NAc) via increases in plas-
malemmal and vesicular dopaminergic uptake (Niwa et al., 2007).
Moreover, we generated Shati/Nat8l-knockout (Shati/Nat8l−/−) mice
on a C57/B6 background (Furukawa-Hibi et al., 2012) which showed
higher dopamine turnover and basal levels of extracellular dopamine
in the NAc (Toriumi et al., 2015). The lack of Shati/Nat8l decreases
both dendrite complexity and leng th in the prefrontal cortex (PFC)
pyramidal neurons (Toriumi et al., 2013). Alternatively, Shati/Nat8l
induces axon outgrowth by adenosine triphosphate synthesis in
the hippocampus (Sumi et al., 2015). The dopamine release after
methamphetamine treatment is also higher in the NAc of Shati/
Nat8l−/− mice (Toriumi et al., 2015). Conversely, overexpression of
Shati/Nat8l in the NAc suppresses the increase in dopamine release
caused by methamphetamine via metabotropic glutamate receptor 3
(Miyamoto et al., 2014). Overexpression of Shati/Nat8l in the medial
PFC attenuates methamphetamine-induced conditioned place pref-
erence and dopamine increases in the NAc (Haddar, Uno, Azuma,
Mura mat su, & Nitt a, 2019). Thes e res ul t s su g ge st th at Sh at i/Nat 8l−/−
mice have dopaminergic abnormalities and that Shati/Nat8l is im-
portant in the regulation of the dopaminergic system.
Currently, the role of Shati/Nat8l in the morphology and adult
neurogenesis in the DG and higher-order brain function remains
largely unknown. Therefore, in the present study, we investigated
the role of Shati/Nat8l in adult neurogenesis and attention via do-
paminergic transmission using Shati/Nat8l−/− mice. We found that
Shati/Nat8l is important in morphogenesis and adult dopaminergic
neurogenesis in the DG and attention through modulating dopamine
transmission.
2 | MATERIALS AND METHODS
2.1 | Animals
The generation of Shati/Nat8l−/− mice has been previously de-
scribed in which Shati/Nat8l DNA (NM 001001985.3) was absent in
C57BL/6J genetic background mice (Furukawa-Hibi et al., 2012; Niwa
et al., 2007) (Research Resource Identifiers, RRID:MGI:5488963).
Male and female (1:1) heterozygous mutant mice were bred to gen-
erate homozygous mutant (Shati/Nat8l−/−) mice (n = 125) and their
wild type (WT; n = 127) littermates. All behavioral experiments were
performed using 8-weeks-old mice during the facility light cycle.
Both male and female mice were used in the current study because
there were no gender differences observed in our previous study
(Fur ukawa-Hi bi et al. , 201 2). The graphical time-lin e of the st udy de-
sign is shown in Figure 1a. This study was not pre-registered and no
randomization/blinding was per formed. No exclusion criteria were
pre-determined, and no animals were excluded. Sample size for each
experiment was determined based on our previous studies with the
relevant type of experiment (Alkam et al., 2011; Nagai et al., 2007;
Sobue et al., 2018; Wulaer et al., 2018). No sample size calculations
were performed. Animals were housed in the animal facilities at the
Fujita Health University Graduate School of Medicine under spe-
cific pathogen-free conditions, maint ained at 25°C on a 12 hr light/
Grant /Award Number: 17H04252,
18K15377, 18K19761 and 19K07490;
Ministry of Education, Cultu re, Sports ,
Science , and Technology of Ja pan; Smo king
Research Foundation; Fujita Heal th
University
KEYWORDS
attention, dendate gyrus, dopamine, morphogenesis, neurogenesis, Shati/Nat8l
  
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WULA ER Et AL.
dark cycle (lights on at 08:00 hours), and had free access to food and
water. All animal care and use were in accordance with the National
Institute of Health Guide for the Care and Use of Laboratory Animals
and were approved by the Animal Experimentation Committee of
Fujita Health University Graduate School of Medicine (Permit
Number: AP1604 4).
2.2 | Golgi staining and morphological analyses
Golgi staining was performed using the FD Rapid Golgi Stain Kit
(Cat# FNT-PK401-1KIT) according to the manufacturer's protocol
(FD NeuroTechnologies) and a previous study (Sobue et al., 2018).
Mice were sacrificed by decapitation then the freshly dissected
brains were immersed in solutions A and B for 1 week at room tem-
perature and transferred to solution C for 24 hr at 4°C. The brains
then sectioned using a cryostat at a thickness of 80 µm. BZ9000
bright-field microscopic (KEYENCE) images of DG neurons were
obtained. Only fully impregnated neurons displaying dendritic trees
without obvious truncations and isolated from neighboring impreg-
nated neurons were retained for analyses. All dendrites within im-
ages were traced using Neurolucida software (RRID:SCR _001775;
MicroBrightField Bioscience) and analyzed using NeuroExplorer
(MicroBrightField). Spine densit y was measured in a range of den-
drites 30–120 μm from the soma. It is expressed as the number of
spines per micrometer of dendritic length. At least three neurons in
each mouse were counted for the analysis.
2.3 | Nissl staining
Nissl staining was performed to measure the thickness in hippocam-
pal subregions. Mice were deeply anesthetized with isoflurane
(8.131 mol/L; Cat# 099-06571; Wako). Once reflex responses had
FIGURE 1 Abnormal morphogenesis
and spinogenesis in the dentate gyrus
(DG) of Shati/Nat8l−/− mice. (a) A
graphical time-line of the study design.
(b) Representative images of DG neurons
in WT and Shati/Nat8l−/− mice. Scale
bars indicate 50 μm. (c) Total dendritic
length. (d) Dendritic length. (e) Dendrite
intersection. (n = 18 dendrites from 6
WT mice; n = 21 dendrites from 7 Shati/
Nat8l−/− mice). (f) Representative images
of dendritic spines from DG neurons in
WT and Shati/Nat8l−/− mice. Scale bars
indicate 5 μm. (g) Spine density (n = 12
neurons from 4 mice per genotype).
*p < .05; **p < .01. All data are expressed
as means ± SEM.
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disappeared, mice were transcardially per fused with 4% paraform-
aldehyde in phosphate buffered saline (PBS). Brains were post-fixed
overnight in 4% paraformaldehyde and cryoprotected in 30% su-
crose in PBS. Sections were cut at 30 µm inter vals, and staining
was done according to the standard procedure (Murai et al., 2007).
Images were acquired with a light microscope (BZ9000; KE YENCE)
and analyzed using ImageJ software (RRID:SCR_003070; National
Institute of Mental Health).
2.4 | Immunofluorescence staining
Immunofluorescence staining was described previously (Wulaer
et al., 2018). Mice were deeply anesthetized with isoflurane (1 ml/
ml; Cat# 099-06571; Wako) and transcardially perfused with 4%
paraformaldehyde in PBS. Brains were post-fixed overnight in 4%
paraformaldehyde and cryoprotected in 30% sucrose in PBS. The
brains were prepared (20 μm) using a cryostat (RRID:SCR_016844;
Leica CM3050 S Research Cryostat).
The coronal sections between −1.46 and −2.18 mm from bregma
(according to the mouse brain atlas) (Paxinos & Franklin, 2004) were
washed with PBS containing 0.3% Triton X-100, incubated at 25°C
for 2 hr in 5% fetal bovine serum (Cat# 172012; Nichirei Biosciences
Inc.), and then incubated with primary antibodies at 4°C overnight.
The information on the antibodies are described in Table 1. After
washing in PBS, secondary antibodies were added to the sec-
tions at 25°C for 1 hr. After washing in PBS, these sections were
mounted on slides and visualized under a confocal laser microscope
(RRID:SCR_013672; Zeiss L SM-710FSX100; Olympus). The number
or density of cells positive for immunoreactivities were analyzed
using ImageJ sof tware. Averages of at least three slices in each
mouse were counted within areas of interest (360 µm × 260 μm) and
used for statistical analysis.
2.5 | 5-bromo-2'-deoxyuridine (BrdU) labeling
One hundred mg/kg BrdU (RRID:AB_305426; Abcam) was given
intraperitoneally (i.p.) in a volume of 0.1 ml/10 g body weight. To
analyze cell proliferation, mice were sacrificed 2 hr after a single
injection of BrdU (Figure 2a,b). To analyze cell survival and cell
type, mice were given four injections of BrdU (once daily for four
con se cu ti ve days) and sacrifi ced af te r 4 weeks (Uchida et al ., 2011).
Mice were deeply anesthetized wit h isoflurane (1 ml/ml; Cat# 099-
06571; Wako). Once reflex responses had disappeared, mice were
transcardially perfused with 4% paraformaldehyde in PBS. Brains
were post-fixed overnight in 4% paraformaldehyde and cryopro-
tected in 30% sucrose in PBS then proceed to the immunofluores-
cence staining.
2.6 | Chamber for object-based attention test
(O BAT )
The chamber was a rectangular, two-chambered, opaque plexiglass
box including an exploring chamber (40 cm × 40 cm × 22 cm) and
test chamber (40 cm × 20 cm × 22 cm). Dividing walls were made
from opaque plexiglass (the dividing wall) with sliding openings that
allowed access between each chamber.
2.7 | O BAT
The task was previously established and described (Alkam
et al., 2011). Briefly, mice were allowed to explore both training and
testing chambers for a total of 10 min for the habituation period.
In the training session, mice were exposed to five similar-sized but
different shaped objects (e.g., objects a-e in Figure 5a) for 3 min.
Name RRID Catalog # Dilution Source
Rat Anti-BrdU AB_305426 ab6326 1:200 Abcam
Mouse Anti-NeuN AB_281494 8 MAB377 1:1,000 Sigma-Aldrich
Mouse Anti-GFAP AB_2827276 S206A−8 1:500 Sigma-Aldrich
Mouse Anti-DCX AB_10 610966 S C−2 7139 0 1:200 Santa Cruz
Hoechst KU039 KU039 1:1,000 Dojindo
Ra bb it-SO X2 AB_2286686 AB5603 1:200 Millipore
Mouse TH AB_297840 ab11 2 1:1,000 Abcam
Donkey anti-rat AB_2535794 A 21208 1:1,000 Thermo Fisher
Scientific
Rabbit anti-mouse AB_27575 47 A10027 1:1,000 Thermo Fisher
Scientific
Goat anti-rabbit AB_2813889 A31632 1:1,000 Thermo Fisher
Scientific
Abbreviations: BrdU, 5-bromo-2'-deox yuridine; DCX, doublecor tin; GFAP, glial fibrillar y acidic
protein; NeuN, neuronal nuclei; RRID, Research Resource Identifier (see scicrunch.org); SOX2, SRY-
box-containing gene 2; TH, tyrosine hydroxylase.
TABLE 1 List of primary antibodies
used in the present study
  
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WULA ER Et AL.
Previous studies have observed that a mouse would spend an equal
amount of time on any of the five objects during a 3 min exposure
(Alkam et al., 2011). Therefore, the time spent in t wo randomly se-
lected objects out of the five was recorded. Mice were immediately
moved to the test session with a familiar and a novel object (e.g.,
object f) was introduced. The recognition index was expressed as
the ratio ( Tf × 100)/(Ta + Tf), where Ta and Tf are the time spent in
the testing session on objec t a and object f, respectively.
FIGURE 2 Impaired neurogenesis and neuronal maturation in the dentate gyrus (DG) of Shati/Nat8l−/− mice. (a) Single BrdU (100 mg/kg.
i.p.) was given 2 hr before mice were sampled to examine the effects of Shati deficiency on cell proliferation. (b) Representative images of
the DG by BrdU (green) and Hoechst (blue) staining in WT and Shati/Nat8l−/− mice. (c) Quantification of BrdU-positive cells in the DG (n = 18
slices from 6 WT mice; n = 15 slices from 5 Shati/Nat8l−/− mice). (d) Representative images of the DG by BrdU (green), NeuN (red, upper
panel), glial fibrillary acidic protein (GFAP) (red, lower panel), and Hoechst (blue) staining in WT and Shati/Nat8l−/− mice. (e) BrdU was given
once per day for 4 days before mice were sampled to examine the effec ts of Shati deficiency on cell survival 30 days later. (f) The graph
shows the numbers of BrdU neurons (NeuN) and astrocytes (GFAP; n = 18 slices from 6 WT mice; n = 15 slices from 5 Shati/Nat8l−/− mice).
(g) Representative images of doublecortin (DCX) (green) staining in WT and Shati/Nat8l−/− mice. (h) The densit y of DCX-positive cells (n = 18
slices from 6 WT mice; n = 18 slices from 6 Shati/Nat8l−/− mice). (i) Representative images of SRY-box-containing gene 2 (SOX2) (red) and
Hoechst (blue) staining in W T and Shati/Nat8l−/− mice. (j) Quantification of SOX2-positive cells (n = 15 slices from 5 W T mice; n = 15 slices
from 5 Shati/Nat8l−/− mice). (k) Representative images of TH (red) staining in W T and Shati/Nat8l−/− mice. (l) The densit y of TH-positive cells
(n = 15 slices from 5 WT mice; n = 15 slices from 5 Shati/Nat8l−/− mice). Scale bar indicates 100 µm. *p < .05; **p < .01. All data are expressed
as means ± SEM.
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2.8 | Drug treatment
Galantamine (GAL; 1.0 mg/k g; Cat# G0293; Tokyo Chemical Industry
Co. Ltd.) (Koseki et al., 2014), a ni cotin ic acety lcho li ne receptor all os-
teric modulator and cholinesterase inhibitor, was dissolved in saline
and orally administered 60 min before the OBAT. To examine the
role of D1, D2, and α7 nicotinic acetylcholine receptor (nAChR) an-
tagonists on the ameliorating effects of GAL on attentional deficits
of Shati/Nat8l−/− mice in the OBAT, SCH23390 (SCH; 0.03 mg/k g;
i.p.; Cat# D054; Sigma-Aldrich) (Nagai et al., 2009), and raclopride
(RAC; 0.3 g/kg; i.p.; Cat# 84225-95-6; Tokyo Chemical Industry Co.
Ltd.) (Nagai et al., 2009) were dissolved in distilled water, methyl ly-
caconitine (MLA; 2 mg/kg; subcutaneous injection; Cat# ab120072;
Sigma-Aldrich) (Mizoguchi et al., 20 09) was dissolved in saline and
were administered 30 min before the OBAT.
2.9| In vivo microdialysis
To measure dopamine release, mice were anesthetized with a mix-
ture of anesthetic, muscle relaxant, analgesic, and sedative such
as medetomidine (0.3 mg/kg; Domitor®; Nippon Zenyaku Kogyo),
butorphanol (5.0 mg/kg; Vetorphale®; Meiji Seika Pharma), mida-
zolam (4.0 mg/kg; Midazolam Sandoz®; Sandoz) to minimize the
pain and reversed by atipamezole (0.15 mg/kg; Antisedan®; Nippon
Zenyaku Kogyo) after completed the surgery. A guide cannula (AG-
6; Eicom) with dummy cannula (AD-6; Eicom) was implanted into
the hippocampus (AP −3.05 mm, ML +0.8 mm, DV −2.0 mm) (Wang
et al., 2007). One day after the operation, a dialysis probe (A-I-6-
2, total length 6 mm; membrane length 2 mm; diameter 0.22 mm;
dead volume 0.368 μL; Eicom) was inserted through the guide c an-
nula and perfused with artificial cerebrospinal fluid (147 mM NaCl,
4 mM KCl, and 2.3 mM CaCl2) at a flow rate of 1 μL/min. The probe
efficiency is 7.2%. Outflow fractions were collected every 10 min.
After the collection of three stable baseline fractions, GAL (1.0 mg/
kg; i.p.) was dissolved in saline and orally administered and the di-
alysates (10 µl) were collected every 10 min for 120 min. Dialysates
were analyzed by high performance liquid chromatography with an
electrochemical detector (Eicom). Three time points were chosen
for measurements to establish baseline dopamine levels. The lowest
detection limit is about 30 fg by using the volume of dialysate, 10 µl
(Max 40 µl; Eicom). Dopamine level was calculated from the data of
chromatogram. Its release was assessed as the percentage of basal
levels. The wave of dopamine in chromatograms was identified by
retention time of that in standard solution, and the concentration
was calculated according to the area of the wave in the solution.
2.10| Real-time quantitative reverse transcription
polymerase chain reaction (qPCR)
Total RNA was isolated using a NucleoSpin® RNA kit (Takara) ac-
cording to the manufacturer's instructions. Mice were sacrificed
by decapitation. All PCR primers were purchased from Integrated
DNA Technologies. The first strand of cDNA was synthesized using
the ReverTra Ace qPCR-RT kit (Toyobo). For the quantitative PCR,
SsoAdvancedTM Universal Probes Supermix (Bio-Rad) was used and
th en su b jecte d to real -tim e PCR qu anti f i c atio n us ing a St epOne Re a l-
Tim e PC R Sys te m (A pp lied Bios ys tems). The qua ntitati ve PCR anal y-
sis was performed using a StepOne analy zer (Life Technologies).
The PCR reaction program consisted of 40 cycles of denaturation
at 95°C for 30 s and annealing and elongation at 60°C for 1 min. To
discriminate specific amplification from non-specific amplification,
melting curve analyses were performed after each PCR reaction.
β-actin was used as a housekeeping gene to normalize all PCR dat a.
Primers used in this study were α7 nAChR (Mm.PT.58.8458406), α4
nAChR (Mm.PT.58.31947103), β2 nAChR (Mm.PT.58.5455853), and
β-actin (Mm.PT.39a.22214843).
2.11 | Data analyses
All data are expressed as means ± standard error of the mean.
Statistical analyses were performed with GraphPad Prism 6.0
(GraphPad Software, Inc.). Significance was assessed using t tests, or
in the case of multiple comparisons, an analysis of variance (ANOVA)
with repeated measures. The Tukey–Kramer test was used for post-
hoc analyses when F ratios were significant. An assessment of the
normality of the data prior to the statistical comparisons has not
been carried out; however, this type of analysis is resistant to devia-
tions from the assumptions of the traditional ordinary-least-squares
ANOVA, and is robust with outliers, thus being insensitive to distri-
butional assumptions (such as normality) (Huber, 1996). No test for
outliers was conducted. The criterion for a significant difference was
p < .01, p < .05 for all statistical evaluation.
3 | RESULTS
3.1 | Abnormal morphogenesis and spinogenesis in
the DG of Shati/Nat8l−/− mice
It is known that pathological conditions cause abnormalities in den-
dritic morphology (Sousa & Almeida, 2012). Therefore, our first aim
was to investigate the role of Shati/Nat8l in the dendritic length
and arborization in DG neurons, we first per formed Golgi staining
(Figure 1b). The total dendritic length in Shati/Nat8l−/− mice was
significantly shorter compared with that in WT mice (t(11) = 2.46,
p < .05; Figure 1c). Dendritic branching was examined using Sholl-
analysis in WT and Shati/Nat8l−/− neurons. Dendrites of Shati/
Nat8l−/− mice showed significantly fewer intersections per Sholl-
segment, in particular those 10–120 μm from the soma, compared
to WT mice (genotype, F(1, 492) = 30.14, p < .01; intersection, F(11,
492) = 12.02, p < .01; genotype × intersection, F(11, 492) = 1.0 0,
p = .44; Figure 1d). Shati/Nat8l−/− mice exhibited shorter dendrite
length (genotype, F(1, 492) = 31.14, p < .01 ; length, F(11, 492) = 17.06,
  
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WULA ER Et AL.
p < .01; genotype × length, F(11, 492) = 0.93, p = .44; Figure 1e). We
also analy zed the density of dendritic spines of the DG neurons in
the Shati/Nat8l−/− mice (Figure 1f). Results revealed that dendritic
spine density in DG neurons was significantly decreased in the mice
(t(6) = 4.49, p < .01; Figure 1g), suggesting that the Shati/Nat8l posi-
tively regulates spine density. Conversely, Nissl staining showed no
obvious changes in the thickness of hippocampal subfields with
comparable numbers of neurons between the subfields (Figures S1
and S2). Thus, the delet ion of Shati/Nat8l altered neuronal mo rphol-
ogy in the DG.
3.2 | Impaired neurogenesis and neuronal
maturation in the DG of Shati/Nat8l−/− mice
To examine the role of Shati/Nat8l in the adult hippocampal neuro-
genesis, we performed BrdU staining (Figure 2). To analyze cell pro-
liferation, brain samples were collec ted 2 hr after a single injection
of BrdU (Figure 2a,b). Compared to WT mice, Shati/Nat8l−/− mice
exhibited significantly fewer BrdU-positive cells in the DG 2 hr after
BrdU injection (t(9) = 5.80, p < .01; Figure 2c). To anal yze ce ll sur vival
and type, mice were given four injections of BrdU (once daily for 4
consecutive days) and brain samples were collected 30 days after
BrdU labeling (Figure 2d,e). The number of BrdU-positive cells was
sig nificantly lowe r in Shati/Nat8l−/− mi ce (genot ype, F(1, 18) = 32.28,
p < .01; cell type, F(1, 18) = 210.5, p < .01; genotype × cell type, F(1,
18) = 29.35, p < .01; Figure 2f) than WT mice. Most BrdU-positive
cells were expressed in the neurons (NeuN), rather than the astro-
cy te s (gli al fib ri ll ar y acidic pro tein [GFAP]; Figu re 2f). To ex amine the
role of Shati/Nat8l in neuronal maturation, we examined the density
of doublecor tin (DCX; a premature neuronal marker)-positive cells in
t h e DG o f S ha ti /N at 8l −/− m ic e (F ig ur e 2g ). W e fo un d th at t he d en si ty o f
DCX-positive cells was greate r in the Shati/Nat8l−/− mi ce (t(9) = 2.52,
p < .05 ; Fig ur e 3h) . We als o ass e ss e d the numb er of SRY-bo x con tai n-
ing gene 2 (SOX2; a neuronal stem cell marker)-positive cells in the
DG of Shati/Nat8l−/− mice (Figure 2i), but found no differences be-
tween the genotypes (t(9) = 0.01, p = .68; Figure 2j). Several studies
suggest that dopamine controls differential steps of adult hippocam-
pal neurogenesis (Baker et al., 20 04; Borta & Hoglinger, 2007). As
Shati/Nat8l−/− mice have showed abnormal dopamine system previ-
ously (Toriumi et al., 2015, 2018), we hypothesized that the impaired
neurogenesis might be developed by abnormal dopaminergic neu-
ronal function in these mice. Accordingly, we analyzed the density
of tyrosine hydroxylase (TH), a dopaminergic marker in the DG of
Shati/Nat8l−/− mice (Figure 2k). The density of TH-positive cells was
significantly decreased in the DG of Shati/Nat8l−/− mice compared
to that in the WT mice (t(8) = 3.18, p < .05; Figure 2l). These results
suggest that the deletion of Shati/Nat8l impairs neurogenesis and
inhibits neuronal maturation in the DG, possibly bec ause of the re-
duction of dopamine level.
3.3 | Impaired neurogenesis of the DG receiving
projection from dopaminergic neurons in Shati/
Nat8l−/− mice
As the dopamine level was significantly decreased in the DG of
Shati/Nat8l−/− mice, we hypothesized that the TH is involved in pro-
liferation an d cell surv ival rate in the DG of adul t Shati/Nat8l−/− mice
and we co-stained using BrdU with TH (Figure 3a). Brain samples
FIGURE 3 Impaired neurogenesis of the dent ate gyrus (DG) receiving the projec tion from dopaminergic neurons in Shati/Nat8l−/− mice.
(a) Represent ative images of the DG by BrdU (green), TH (red), and Hoechst (blue) staining in WT and Shati/Nat8l−/− mice. (b) Single BrdU
(100 mg/kg. i.p.) was given 2 hr before mice were sampled to examine the effects of Shati deficiency on dopaminergic cell proliferation. (c)
Percentage of BrdU-positive cells in the TH-positive cells in the DG . Scale bar indicates 100 µm. (n = 18 slices from 6 WT mice; n = 18 slices
from 6 Shati/Nat8l−/− mice). *p < .05. All data are expressed as means ± SEM.
8 
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   WULAER Et AL.
were collected 2 hr after a single BrdU injection (Figure 3b). The
percentage of BrdU-positive cells surrounded with TH-positive fib-
ers was significantly lower in the Shati/Nat8l−/− mice compared to
the WT mice (t(9) = 2.72, p < .05; Figure 3c). Fur thermore, SOX2
with BrdU and DCX with BrdU were used as co-stains to investigate
whether progenitor cells have impairment s in proliferation or if there
is a change in newborn immature neurons, respectively (Figure S3a–
d). However, data showed no differences bet ween the genotypes.
These results indicate that the deletion of Shati/Nat8l reduces neu-
rogenesis of the DG, which receives projection from dopaminergic
neurons.
3.4 | Deceased survival rate in newborn cells in the
DG receiving projection from dopaminergic neurons
in Shati/Nat8l−/− mice
To examine the sur vival rate of newborn cells in the DG that receive
projection from dopaminergic neurons in adult Shati/Nat8l−/− mice,
we co-stained BrdU with TH (Figure 4a) in a different experimen-
tal schedule. BrdU injections were given four times daily, then brain
samples were collected 30 days later (Figure 4b). The percentage
of BrdU-positive cells surrounded by TH-positive fibers were sig-
nificantly lower in the Shati/Nat8l−/− mice compared to the WT
mice (t(9) = 5.71, p < .01; Figure 4c). Next, we co-stained BrdU with
DCX to investigate whether the BrdU cells have committed towards
a neurogenic fate, however there was no difference between the
genotypes (Figure S3f–h). These results suggest that the deletion of
Shati/Nat8l reduces the survival rate of newborn cells in the DG that
receive projection from dopaminergic neurons, but not their neuro-
genic fate.
3.5 | GAL ameliorated attentional impairment
in the OBAT and dopaminergic dysfunction in the
hippocampus of Shati/Nat8l−/− mice
The hippocampus is brain region implicated in the regulation of at-
tention (Chudasama, Doobay, & Liu, 2012). Normal performance
in attention-related tasks require intact adult hippocampal neuro-
genesis (Eadie et al., 2009; Moon et al., 2006). Previously, on the
OBAT, Shati/Nat8l−/− mice demonstrated a notably impaired perfor-
mance (Toriumi et al., 2018). Therefore, we examined the effects
of GAL , nAChR-allosteric modulator, and a modest cholinesterase
inhibitor (intended to improve cognition in attention-deficit hyper-
activity disorder patients (Eisele et al., 1993; Santos et al., 2002;
Bracco, Bessi, Padiglioni, Marini, & Pepeu, 2014)) on attentional im-
pairments in Shati/Nat8l−/− mice during the OBAT (Figure 5). In a
3 min training session, genotypes showed no differences with any
treatments in the exploration time to object a or b (F(5, 84) = 0.50,
p = .77; F(5, 84) = 0.45, p = .81; Figure 5a). After a 10 s interval,
mice were moved to the test session with the familiar object, and
a novel object was introduced. The Shati/Nat8l−/− mice showed a
reduction of recognition index, which was ameliorated with GAL
administration (Figure 5b-c). To examine the involvement of α7
nAChR in the ameliorating effects of GAL, Shati/Nat8l−/− mice
were co-administered GAL with MLA (an α7 nAChR antagonist).
The ameliorating effect of GAL was blocked by the MLA in Shati/
FIGURE 4 Deceased survival rate in newborn cells of the dentate gyrus (DG) receiving the projection from dopaminergic neurons in
Shati/Nat8l−/− mice. (a) Representative images of the DG in BrdU (green), TH (red), and Hoechst (blue) staining in WT and Shati/Nat8l−/−
mice. (b) BrdU was given once per day for 4 days before mice were sampled to examine the ef fect s of Shati deficiency on dopaminergic cell
survival 30 days later. (c) Percentage of BrdU-positive cells in the TH-positive cells in the DG. Scale bar indicates 100 µm. (n = 18 slices from
6 WT mice; n = 18 slices from 6 Shati/Nat8l−/− mice). **p < .01. All data are expressed as means ± SEM.
  
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WULA ER Et AL.
Nat8l−/− mice (F(5, 83) = 27.70, p < .01; Figure 5b-c); however, the
gene expression of nAChR subtypes (α7, α4, β2) was not af fected
by either Shati/Nat8l deficiency or GAL treatment (Figure S4). The
density of TH-positive cells was significantly lower in the DG of
Shati/Nat8l−/− mice (Figure 2kl) and the survival rate of newborn
cells of the DG receiving projection from dopaminergic neurons in
adult Shati/Nat8l−/− mi ce was reduced (Figur e 4c). Therefore, we hy-
pothesized that the dopaminergic neuronal system in the hippocam-
pus was impaired in Shati/Nat8l−/− mice. As shown in Figure 5d, the
basal extracellular level of dopamine in the hippocampus of Shati/
FIGURE 5 Galantamine (GAL) ameliorated attentional impairment in objec t-based attention test and dopaminergic dysfunction in the
hippocampus of Shati/Nat8l−/− mice. (a) Mice were exposed to five objects for 3 min (training session), then, within an interval of 10 s, they
were exposed to two objects that include a familiar and a novel object for 3 min (test session). (b) Object exploration time in the training
session. (c). Recognition index in the test session (WT Saline n = 14 mice, WT GAL n = 15 mice, W T methyllycaconitine (MLA) n = 16 mice,
Shati/Nat8l−/− Saline n = 16 mice, Shati/Nat8l−/− GAL n = 17 mice, Shati/Nat8l−/− MLA n = 11 mice). (d) Dopamine basal level. (e) Percentage
of dopamine basal level, the arrow indicates injec tion time of GAL (WT n = 5 mice, Shati/Nat8l−/− n = 5 mice). (f) Object exploration time
in the training session. (g). Recognition index in the test session (n = 11 mice/group). GAL, 1.0 mg/k g, p.o.; ML A 2 mg/kg; subcutaneous
injection; SCH, SCH 23390, 0.03 mg/kg; i.p; R AC, raclopride, 0.3 g/kg; i.p. *p < .05, **p < .01. All data are expressed as means ± SEM.
10 
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   WULAER Et AL.
Nat8l−/− mice was significantly decreased compared to that of WT
mice (t(8) = 2.56, p < .05; Figure 5d). Previously, we have reported
that GAL ameliorates cognitive dysfunction by facilitating the in-
crease in the extracellular level of dopamine release in the hip-
pocampus of mice (Wang et al., 2007). We performed microdialysis
to examine whether GAL ameliorated the attention deficits through
facilitating dopamine release in the hippocampus. The lowered ex-
tracellular level of dopamine in Shati/Nat8l−/− mice was markedly
in cr e as e d by a sing le in jec tion of GAL (1 mg/ k g), wher eas ther e wer e
no changes obser ved in W T mice (F(5, 6) = 5.297, p < .05; Figure 5e).
To clarify the involvement of dopaminergic subtypes in the ame-
liorating effects of GAL, Shati/Nat8l−/− mice were co-administered
GAL with SCH23390 (SCH; a dopamine D1 receptor antagonist) or
racl op ride (R AC ; a dop am in e D2 receptor ant ag on is t). In th e trainin g
session, mice showed no genotypic differences in the exploration
time with objects a or b (F(6, 70) = 0.30, p = .93; F(6, 70) = 0.21,
p = .97; Figure 5f). In the test session, the reduced recognition index
in the Shati/Nat8l−/− mice was improved with GAL treatment and
th is im p rov eme nt wa s bloc ked by SCH bu t not RAC (F(6 , 70) = 12 . 22,
p < .01; Figure 5g). These results indicate that attentional impair-
ments observed in the OBAT in Shati/Nat8l−/− mice was because
of hypodopaminergic function in the hippocampus; however, GAL
ameliorated attentional impairments possibly by activating D1 re-
ceptors following an increase in dopamine release via the α7 nAChR
in the hippocampus.
4 | DISCUSSION
Here we investigated the effects of genetic Shati/Nat8l deficiency
on adult neurogenesis and attention. We found that the genetic
deletion of Shati/Nat8l reduced hippocampal dendrite complexity,
shortened dendrite length, decreased proliferation, decreased sur-
vival rate of newborn cells in the DG receiving the projection from
dopaminergic neurons, increased premature neurons, and impaired
dopaminergic function in the hippocampus as well as attention in
the OBAT. These results suggest that Shati/Nat8l is important in
morphogenesis and adult neurogenesis in the DG and at tention via
modulation of dopamine transmission.
In this study, Shati/Nat8l deletion caused alterations in the
morphology of DG neurons which were evidenced by the reduced
dendrite length and complexity as well as spine density (Figure 1). It
is known that the connectivity of the neuronal network is strongly
determined by its dendritic–axonal arborization and synaptic con-
tacts (van Pelt & van Ooyen, 2013). The Sholl-analysis revealed a
decrease in the complexity of neurons in the DG which suggest s that
deletion of Shati/Nat8l induces changes in the morpholog y of neu-
rons and results in decreased connectivity within the neural circuit.
The DG is characterized by the presence of neuronal progenitor cells
throughout the entire life of mammals. Neural precursor cells that
reside in the DG proliferate, survive, and dif ferentiate into the neu-
ron and/or glia, then begin maturation (Kempermann, 2006; Ming
& Song, 2005). Here, progenitor proliferation and survival affected
the neuronal cells as compared to the glial cells (Figure 2f). There
was no dif ference in the number of SOX2-positive cells, while the
density of DCX was signific antly increased although the number of
BrdU/NeuN-positive mature neurons were significantly decreased
in the Shati/Nat8l−/− mice (F igure 2). The Shati/Nat8l−/− mice showed
no differences in proliferation of BrdU/DCX-positive newborn and
BrdU/SOX2-positive immature neurons (Figure S3). It is a very
surprising and interesting finding in Shati/Nat8l−/− mice. Previous
reports have shown that removal of polysialic acid-neural cell adhe-
sion molecule (PSA-NC AM), a late progenitor and immature neuron
marker, enhances neuronal differentiation but inhibits synapse for-
mation in adult hippocampal neurons (Burgess et al., 2008; Dityatev
et al., 2004), this supports our observations of increased density of
DCX and altered dendritic morphologies in the DG of Shati/Nat8l−/−
mice. One possible reason for this phenomenon is that Shati/Nat8l
may interact with PSA-NCAM involved in the inhibition of DG neu-
ronal maturation.
In a previous study, increased DCX- and BrdU-positive cells
were found in the DG of an Alzheimer's disease mouse model
by increasing the level of NA A (Zhang et al., 2017). Conversely,
antidepressant treatment increased hippocampal NA A levels
with in patients with major depressive disorder, rescued the im-
paired neurogenesis, and improved cognition (Wang et al., 2012).
Given that the hippocampus is important in attention and adult
hippocampal neurogenesis is required for normal at tentional per-
formance, we suggest that impaired neurogenesis in the DG and
attentional impairments in Shati/Nat8l−/− mice might be because
of reduced NAA levels (Chudasama et al., 2012; Eadie et al., 2009;
Moon et al., 2006). However, further experiments are needed to
confirm this. Alternatively, the integration of adult-generated im-
matu re neurons into the exis ting neuronal circuit of the hippocam-
pus is important for adult hippocampal neurogenesis in cognition,
including at tention (Deng, Aimone, & Gage, 2010). Therefore, the
impaired attention observed in the OBAT in Shati/Nat8l−/− mice
is likely because of the combination of reduced numbers of new
neurons and aberrant integration of immature neu rons into the ex-
isting circuitry.
In our previous studies, we found attention impairment and
decreased dopaminergic turnover in the PFC of Shati/Nat8l−/−
mice (Toriumi et al., 2018). The reduced attentional impairment
was recovered with methylphenidate and atomoxetine treat-
ments, suggesting that attention deficits in Shati/Natl−/− mice are
associated with hypodopaminergic function (Toriumi et al., 2018).
The hippocampus exhibits potent reciprocal connections with the
PFC and these areas receive intense dopaminergic innervation
and are implicated in attention and impulse behavior (Schaefers,
Teuchert-Noodt, Bagorda, & Brummelte, 2009). Therefore, the
DG is under direct and indirect dopaminergic influence from its
afferents. Disturbance to the dopaminergic system may have an
essential impact on hippocampal activity and function (Schaefers
et al., 2009).
Dopamine signaling regulates the proliferation of neural pre-
cursor cells which demonstrates a reduction of mitotic activity in
  
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 11
WULA ER Et AL.
the number of neural precursor cells in the DG of individuals with
Parkinson's disease (Hoglinger et al., 2004). Here we found that the
decreased proliferation and survival rate of newborn cells in the DG
receives projection from dopaminergic neurons in Shati/Nat8l−/−
mice, suggesting that Shati/Nat8l deficiency impairs neurogenesis
and decreases survival rate via hypodopaminergic function in the
hippocampus. A month BrdU labeling method was used to check
the survival of proliferating cells. Although there was a decrease
in BrdU-labeled TH-positive cell count, there is an important lim-
itation that the data does not distinguish whether these BrdU cells
committed towards a neurogenic fate. Dopaminergic fibers in the
DG originate partly from the substantia nigra, but mainly from the
ventral tegmental area (Gasbarri, Sulli, & Packard, 1997). The ventral
tegmental area directly innervates the hippocampus, suggesting a
functional and anatomical connection of dopamine with distal brain
regions (Baker et al., 2004; Hoglinger et al., 2004). Dopaminergic
denervation of the forebrain reduces proliferation in the DG fur-
ther suggesting that the dopaminergic projection from the ventral
tegmental area is one of the key regulators of neurogenesis in the
adult hippocampus (Girault & Greengard, 2004). It is well known
that dopamine is involved in the regulation of motivation, attention,
working memory, and reward (Brown et al., 2010; Phillips, Vacca, &
Ahn, 2008). Therefore, the impaired dopaminergic neurogenesis in
the DG of Shati/Nat8l−/− mice may be caused through projections
from other brain regions including the PFC and leads to behavioral
impairment; however, the adult hippocampal circuit was regulated
by dopamine and multiple modulatory neurotransmitters, such as
acetylcholine and serotonin (Song, Olsen, Sun, Ming, & Song, 2016).
Further studies are needed to clarify the role of Shati/Nat8l defi-
ciency in neurogenesis and attention related to the different neu-
ronal projections (e.g., projection specific, cell-type specific) in
behavioral regulation.
Th e de cre ase d recog nitio n ind ex on the OBAT ob se r ved in Shati /
Nat8l−/− mice were ameliorated by treatment with GAL (Figure 5).
GAL has a dual mechanism of action; it inhibits acetylcholinester-
ase and allosterically modulates nAChR (Eisele et al., 1993; Santos
et al., 2002). Researchers have previously shown that 14 days of
repeated treatment with GAL promotes neurogenesis in the hippo-
campus of mice (Jin, Xie, Mao, & Greenberg, 2006). Alternatively,
a single injection of GAL also promoted the proliferation of newly
divided cells in the DG and this effect was blocked by MLA (Kita
et al., 2014). Similarly, the ameliorating effect of GAL on attentional
deficits in Shati/Nat8l−/− mice was blocked by treatment with MLA
(Figure 5c). These findings support the notion that GAL improves
the attentional impairments in Shati/Nat8l−/− mice via activation
of α7 nAChR. These results are consistent with our earlier report
that the increase of dopamine release in the hippocampus by GAL
is potentiated by nicotine and antagonized by mecamylamine (Wang
et al., 2007).
However, at the mRNA level, none of the subtypes of nAChR
were ch anged by Shat i/Nat 8l def ici en c y o r G AL tr e at m en t (Fig ur e
S4). In addition to nicotinic receptors, we have previously re-
ported that GAL at a dose of 3.0 mg/kg augments dopaminergic
neurotransmission in the hippocampus (Wang et al., 2007).
In the present study, we used GAL at a dose of 1.0 mg/kg be-
cause higher doses of GAL cause hypoactivity in mice (data not
shown). The lowered extracellular level of dopamine in Shati/
Nat8l−/− mice was markedly recovered by a single injection of
GAL, whereas there were no changes observed in the WT mice
(Figure 5d,e). The decreased level of dopamine, observed in the
microdialysis, is consistent with the decreased density of TH in
the DG of Shati/Nat8l−/− mice. Therefore, the reduction in dopa-
mine levels caused by Shati/Nat8l deficiency may directly affect
th e p ro duc tion of new neu ro ns in the DG . T he am eli ora tin g effe ct
of GAL was blocked by SCH, which indicated the participation of
th e D1 re ce pt or. Co nsist ent w it h previo us re por t s, GAL enha nc ed
dopaminergic neurotransmission in vivo via allosteric potentia-
tion of nAChR (Noda et al., 2010; Schilstrom, Ivanov, Wiker, &
Svensson, 2007). Our study has successfully demonstrated that
GAL may improve at tentional impairments in Shati/Nat8l−/− mice
through th e act iv at io n of D1 receptors v ia augmentation of dopa-
minergic release by the allosteric potentiation of α7 nAChR i n th e
DG. Nevertheless, since we have used systematic administration
of GAL to mice, this positive effect may not only limit to DG, but
also to other brain regions in the Shati/Nat8l−/− mice (Furukawa-
Hibi et al., 2012; Toriumi et al., 2015, 2018). Further investiga-
tion of local injection of GAL to DG of Shati/Nat8l−/− DG will be
needed.
In conclusion, Shati/Nat8l is important in attention and the mor-
phogenesis and neurogenesis in the DG. Impaired neurogenesis
might be because of the reduction in dopaminergic function in the
hippocampus. Pharmacological studies suggest that the ameliorating
effect of GAL on lack of Shati/Nat8l-induced attentional impairment
may associated with the activation of D1 receptors following aug-
mentation of dopamine release via the allosteric potentiation of α7
nAChR in the hippocampus.
ACKNOWLEDGEMENTS
This wor k was sup po rte d by Gr an ts-i n-Aids for Scie nt if ic Rese ar ch
from the Japan Society for the Promotion of Science (17H04252,
18K15377, 18K19761, and 19K07490); the Private University
Research Branding Project from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan; and the research grant
from the Smoking Research Foundation. This work was also sup-
ported by the Education and Research Facilit y of Animal Models
for Human Diseases at Fujita Health University. All experiments
were conducted in compliance with the ARRIVE guidelines. The
authors report no biomedical financial interests or potential con-
flicts of interest.
AUTHOR CONTRIBUTIONS
BW devised the project and the main conceptual ideas, participated
in all experiments and drafted the manuscript. KK supervised the
work and drafted the manuscript. KH, WJS, HK, TI, and AK assisted
with experiments. TN and KY provided necessary software for ex-
periments and involved in the manuscript discussion. AN, YY, and KS
12 
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   WULAER Et AL.
contributed to the manuscript discussion. AM and TN supervised the
work and finalized the manuscript.
ORCID
Bolati Wulaer https://orcid.org/0000-0002-0864-9870
Kazuo Kunisawa https://orcid.org/0000-0002-4786-9681
Taku Nagai https://orcid.org/0000-0001-5050-7748
Kuniaki Saito https://orcid.org/0000-0001-5800-9305
Akihiro Mouri https://orcid.org/0000-0003-3833-4041
REFERENCES
Alkam, T., Hiramatsu, M., Mamiya, T., Aoyama, Y., Nitta, A., Yamada, K.,
… Nabeshima, T. (2011). Evaluation of object-based attention in mice.
Behavioral Brain Research, 220, 185–193. ht tps://doi.org/10.1016/j.
bbr.2011.01.039
Ariyannur, P. S., Moffett, J. R., Manickam, P., Pattabiraman, N., Arun,
P., Nitta, A ., Namboodiri, A. M. (2010). Methamphetamine-
induced neuronal protein NAT8L is the NAA biosynthetic enzyme:
Implications for specialized acetyl coenzyme A metabolism in the
CNS. Brain Research, 1335, 1–13. https://doi.org/10.1016/j.brain
res.2010.04.008
Baker, S. A., Baker, K. A., & Hagg, T. (20 04). Dopaminergic nigrostria-
tal projections regulate neural precursor proliferation in t he adult
mouse subventricular zone. European Journal of Neuroscience, 20,
575–579. https ://doi.or g/10.1111/j.14 60 -9568. 20 04.03486.x
Bangasser, D. A., Dong, H., Carroll, J., Plona, Z., Ding, H., Rodriguez, L., …
Valentino, R. J. (2017). Corticotropin-releasing fac tor overexpression
gives rise to sex differences in Alzheimer's disease-related signal-
ing. Molecular Psychiatry, 22, 1126–1133. https://doi.org/10.1038/
mp.2016 .185
Borta, A., & Hoglinger, G . U. (2007). Dopamine and adult neuro-
genesis. Journal of Neurochemistry, 100, 587–595. https://doi.
org /10.1111/j .1471-4159.20 06.04241.x
Bracco, L ., Be ssi, V., Padiglio ni, S., Marini , S., & Pepeu, G. (2014). Do cho-
linesterase inhibitors ac t primarily on attention deficit? A naturalistic
study in Alzheimer's disease patients. Journal of Alzheimer's Disease,
40, 737–742. https://doi.org/10.3233/JAD-131154
Brown, J. A ., Emnett, R. J., White, C. R., Yuede, C. M., Conyers, S. B .,
O'Malley, K. L., … G utmann, D. H. (2010). Reduced striat al dopamine
underlies the attention system dysfunction in neurofibromatosis-1
mutant mice. Human Molecular Genetics, 19, 4515–4528. https://doi.
org/10.1093/hmg/ddq382
Burgess, A., Wainwright, S. R., Shihabuddin, L. S., Rutishauser, U., Seki,
T., & Aubert , I. (20 08). Polysialic acid regulates the clustering, migra-
tion, and neuronal differentiation of progenitor cells in the adult hip-
pocampus. Dev Neurobiol, 68, 1580–1590. https://doi.org/10.1002/
dneu.20681
Chudasama, Y., Doobay, V. M., & Liu, Y. (2012). Hippocampal-prefrontal
cortical circuit mediates inhibitory response control in the rat.
Journal of Neuroscience, 32, 10915–10924. https://doi.or g/10.1 523/
JNEUR OSCI.1463-12.2012
Deng, W., A imone, J. B., & Gage, F. H. (2010). New neurons and new
memories: How does adult hippocampal neurogenesis affect learn-
ing and memory? Nature Reviews Neuroscience, 11, 339–350. https://
doi.org/10.10 38/nr n2822
Dioli, C., Patrício, P., Sousa, N., Kokras, N., Dalla, C., Guerreiro, S., …
Sotiropoulos, I. (2019). Chronic stress triggers divergent dendritic al-
terations in immature neurons of the adult hippocampus, depending
on their ultimate terminal fields. Transl Psychiatry, 9, 143. ht tps://doi.
org /10.1038/s4139 8-019-0477-7
Dioli, C., Patrício, P., Trindade, R., Pinto, L. G., Silva, J. M ., Morais, M.,
Sotiropoulos, I. (2017). Tau-dependent suppression of adult
neurogenesis in the stressed hippocampus. Molecular Psychiatry, 22,
1110–1118. h tt ps ://doi.or g/10.1038/m p. 2017.103
Dityatev, A ., Dityateva, G., Sytnyk, V., Delling, M., Toni, N., Nikonenko,
I., … Schachner, M. (200 4). Polysialylated neural cell adhesion mole-
cule promotes remodeling and formation of hippocampal synapses.
Journal of Neuroscience, 24, 9372–9382. https://doi.org/10.1523/
JNEUR OSCI.1702-04.2004
Eadie, B. D., Zhang, W. N., Boehme, F., Gil-Mohapel, J., Kainer, L.,
Sim ps on , J. M., & Chr is ti e, B. R. (200 9). Fmr1 knockou t mi ce show re-
duced anxiety and alterations in neurogenesis that are specif ic to the
ventral dentate gyrus. Neurobiology of Diseases, 36, 361–373. htt ps ://
doi.org/10.1016/j.nbd.2009.08.001
Eisele, J. L., Bertrand, S., Galzi, J. L., Devillers-Thier y, A., Changeux, J.
P., & Bertrand, D. (1993). Chimaeric nicotinic-serotonergic receptor
combines distinct ligand binding and channel specificities. Nature,
366, 479–483. https://doi.org/10.1038/366479a0
Furukawa-Hibi, Y., Nitta, A., Fukumitsu , H., Somiya, H., Toriumi, K.,
Furukawa, S., … Yamada, K. (2012). Absence of SHATI/Nat8l reduces
social interaction in mice. Neuroscience Letters, 526, 79–84. https://
doi.org/10.1016/j.neulet.2012.08.028
Gasbar ri, A ., Sulli, A., & Packard, M. G. (1997). The dopaminergic mesen-
cephalic projections to the hippocampal formation in the rat. Progress
in Neuro-Psychopharmacology and Biological Psychiatry, 21, 1–22.
h t t p s : / / d o i . o r g / 1 0 . 1 0 1 6 / S 0 2 7 8 - 5 8 4 6 ( 9 6 ) 0 0 1 5 7 - 1
Girault, J. A ., & Greengard, P. (2004). The neurobiology of dopamine sig-
naling. Archives of Neurology, 61, 641–644. https://doi.org/10.1001/
archn eur.61.5.641
Haddar, M., Uno, K., Azuma, K., Muramatsu, S. I., & Nitta, A. (2019).
Inhibitory effects of Shati/Nat8l overexpression in the medial pre-
frontal cortex on methamphetamine-induced conditioned place
preference in mice. Addict Biol. ht tp s://doi.o rg /10.1111 /adb.12749
Hoglinger, G. U., Rizk, P., Muriel, M. P., Duyckaerts, C., Oer tel, W. H.,
Caille, I., & Hirsch, E. C. (2004). Dopamine depletion impairs precur-
sor cell proliferation in Parkinson disease. Nature Neuroscience, 7,
726–735. https://doi.org/10.1038/nn1265
Huber, P. J. (1996) Robust statistic al procedures, Vol. 68: CBMS-NSF
regional conference series in applied mathematics. Society for
Industrial and Applied Mathematics, Philadelphia.
Jin, K., Xie, L., Mao, X. O., & Greenberg, D. A. (2006). Alzheimer's disease
drugs promote neurogenesis. Brain Research, 1085, 18 3–18 8. ht tp s://
doi.org/10.1016/j.brain res.2006.02.081
Kempermann, G. (200 6). Adult neur ogenesis: Stem cells an d neuronal devel-
opment in the adult brain. New York: Oxford University Press.
Kita, Y., Ago, Y., Higashino, K., Asada, K., Takano, E., Takuma , K., &
Matsuda, T. (2014). Galantamine promotes adult hippocampal
neurogenesis via M(1) muscarinic and alpha7 nicotinic receptors
in mice. International Journal of Neuropsychopharmacology, 17,
1957–1968.
Koseki, T., Mouri, A ., Suzuki, S., Nakajima, A ., Mamiya, T., Yan,
Y., & Nabeshima, T. (2014). Galantamine attenuates re-
instatement of cue-induced methamphetamine-seek-
ing behavior in mice. Addiction Biology, 19, 1–4. https://doi.
org /10.1111/j .1369-160 0. 2011.0 0 425 .x
Mahar, I., Bambico, F. R., Mechawar, N., & Nobrega, J. N . (2014). Stress,
serotonin, and hippocampal neurogenesis in relation to depression
and antidepressant effects. Neuroscience and Biobehavioral Reviews,
38, 173–192. https://doi.org/10.1016/j.neubi orev.2013.11.009
Mateus-Pinheiro, A., Pinto, L., Bessa, J. M., Morais, M., Alves, N. D.,
Monteiro, S., … Sous a, N. (2013). Sustained remission from de-
pressive-like behavior depends on hippocampal neurogene-
sis. Translational Psychiatry, 3, e210. https://doi.org/10.10 38/
tp.2 012.141
Ming, G. L., & Song, H. (2005). Adult neurogenesis in t he mammalian
central nervous system. Annual Review of Neuroscience, 28, 223–250.
https://doi.org/10.1146/annur ev.neuro.28.051804.101459
  
|
 13
WULA ER Et AL.
Miyamoto, Y., Ishikawa, Y., Iegaki, N., Sumi, K., Fu, K., Sato, K., … Nitta, A.
(2014). Overexpression of Shati/Nat8l, an N-acetyltransferase, in the
nucleus accumbens attenuates the response to methamphetamine
via activation of group II mGluRs in mice. International Journal of
Neuropsychopharmacology, 17, 1283–1294. https://doi.org/10.1017/
S1461 14571 40 0 011X
Mizoguchi, H., Arai, S., Koike, H., Ibi, D., Kamei, H., Nabeshima, T., …
Yamada, K. (2009). Therapeutic potential of nicotine for metham-
phetamine-induced impairment of sensorimotor gating: Involvement
of pallidotegmental neurons. Psychopharmacology (Berl), 207, 235–
243. https://doi.org/10.1007/s0021 3-009-1651-z
Moon, J., Beaudin, A . E., Verosky, S., Driscoll, L. L., Weiskopf, M.,
Levitsky, D. A., … Strupp, B. J. (2006). Attentional dysfunction, im-
pulsivity, and resistance to change in a mouse model of fragile X
syndrome. Behavioral Neuroscience, 120, 1367–1379. https://doi .
org /10.1037/0735-704 4.120.6.1367
Murai, R., Noda, Y., Matsui, K., Kamei, H., Mouri, A., Matsuba, K., …
Nabeshima, T. (2007). Hypofunctional glutamatergic neurotransmis-
sion in the prefrontal cortex is involved in the emotional deficit in-
duced by repeated treatment with phencyclidine in mice: Implications
for abnormalities of glutamate release and NMDA-CaMKII signaling.
Behavioral Brain Research, 180, 152–160. https://doi.or g/10.1016/j.
bbr.2007.03.003
Nagai, T., Murai, R., Matsui, K., Kamei, H., Noda, Y., Furukawa, H., &
Nabeshima, T. (2009). Aripiprazole ameliorates phencyclidine-in-
duced impairment of recognition memory through dopamine D1 and
serotonin 5-HT1A receptors. Psychopharmacology (Berl), 202, 315–
328. https://doi.org/10.1007/s0021 3-008-1240-6
Nagai, T., Takuma, K., Kamei, H., Ito, Y., Nakamichi, N., Ibi, D., … Yamada,
K. (20 07). Dopam in e D1 receptors reg ul at e pr otei n sy nt he sis-d ep en-
dent long-term recognition memory via extracellular signal-regulated
kinase 1/2 in the prefrontal cortex. Learning and Memory, 14, 11 7–
125. https://doi.org/10.1101/lm.461407
Niwa, M., Nitta, A., Mizoguchi, H., Ito, Y., Noda, Y., Nagai, T., & Nabeshima,
T. (2007). A novel molecule "shati" is involved in methamphet-
amine-induced hyperlocomotion, sensitization, and conditioned
place preference. Journal of Neuroscience, 27, 7604–7615 . ht tps://doi .
org /10.1523/JNEUR OSCI .1575-07.2007
Noda, Y., Mouri, A., Ando, Y. U., Waki, Y., Yamada, S.-N., Yoshimi, A., …
Nabeshima, T. (2010). Galantamine ameliorates the impairment of
recognition memory in mice repeatedly treated with methamphet-
amine: Involvement of allosteric potentiation of nicotinic acetyl-
choline receptors and dopaminergic-ERK1/2 systems. International
Journal of Neuropsychopharmacology, 13, 1343–1354. ht tps://doi.
org /10.1017/S1461 14571 00 0 022 2
Par k , J. H. , & Eni kol o p o v, G . (2 010 ). Tra ns i e n t el evat i o n of ad u l t hip p o c am-
pal neurogenesis after dopamine depletion. Experimental Neurology,
222, 267–276. https://doi.org/10.1016/j.expne urol.2010.01.004
Paxinos, G., & Franklin, K. B. J. (2004). The Mouse Brain in Stereotaxic
Coordinates (2nd ed.). Amsterdam, the Netherlands and Boston, MA:
Elsevier Academic Press.
Phillips , A. G., Vacca, G ., & Ahn, S. (2008). A top-down perspec-
tive on dopamine, motivation and memory. Pharmacology,
Biochemistry and Behavior, 90, 2 36 –249. http s://d oi. org /10. 10 16 /j.
pbb.2007.10.014
Santos, M. D., Alkondon, M., Pereira, E. F., Aracava, Y., Eisenberg, H. M.,
Maelicke, A., & Albuquerque, E. X. (2002). The nicotinic allosteric
potentiating ligand galantamine facilitates synaptic transmission in
the mammalian central nervous system. Molecular Pharmacology, 61,
1222–1234. https://doi.org/10.1124/mol.61.5.1222
Schaefers, A. T., Teucher t-Noodt, G., Bagorda, F., & Brummelte, S.
(2009). Effect of postnatal methamphetamine trauma and adoles-
cent methylphenidate treatment on adult hippocampal neurogenesis
in gerbils. European Journal of Pharmacology, 616 , 86–90. https://doi.
org/10.1016/j.ejphar.2009.06.006
Schilstrom, B., Ivanov, V. B., Wiker, C ., & Svensson, T. H. (20 07).
Galantamine enhances dopaminergic neurotransmission in vivo
via allosteric potentiation of nicotinic acetylcholine receptors.
Neuropsychopharmacology, 32, 4 3–53. ht tps://doi.org /10.103 8/
sj.npp.1301087
So bue , A. , Ito, N. , Nag ai, T., Sh an, W. , Hada , K., Na kaj ima , A. , … Yam a da,
K. (2018). Astroglial major histocompatibility complex class I fol-
lowing immune activation leads to behavioral and neuropatho-
logical changes. Glia, 66, 1034–1052. ht tps ://doi.o rg/10.1002/
glia.23299
Song, J., Olsen, R. H., Sun, J., Ming , G. L., & Song, H. (2016). Neuronal
circuitry mechanisms regulating adult mammalian neurogenesis. Cold
Spring Harb Perspect Biol, 8. https://doi.org/10.1101/cshpe rspect.
a018937
Sousa, N., & Almeida, O. F. (2012). Disconnection and reconnec-
tion: The morphologic al basis of (mal)adaptation to stress.
Trends in Neurosciences, 35, 742–751. ht tp s://doi.org /10.1016/ j.
tins.2012.08.006
Sumi, K., Uno, K., Matsumura, S., Miyamoto, Y., Furukawa-Hibi, Y.,
Muramatsu, S., … Nitta, A. (2015). Induction of neuronal axon out-
growth by Shati/Nat8l by energ y metabolism in mice cultured
neurons. NeuroReport, 26, 74 0–746. ht t ps ://d oi .o rg/10.1 09 7/
WNR.00000 00000 000416
Toriumi, K., Ikami, M., Kondo, M., Mouri, A., Koseki, T., Ibi, D.,
Nabeshima, T. (2013). SHATI/NAT8L regulates neurite outgrowth
via microtubule stabilization. Journal of Neuroscience Research, 91,
1525–1532 . ht tps ://doi.org/10.100 2/j nr.23273
Toriumi, K., Mamiya, T., Song, Z., Honjo, T., Watanabe, H., Tanaka, J.,
… Nabeshima, T. (2015). Deletion of SHATI/NAT8L decreases the
N-acetylaspar tate content in the brain and induces behavioral defi-
cits, which can be ameliorated by administering N-acetylaspartate.
European Neuropsychopharmacology, 25, 2108–2117. https://doi.
org/10.1016/j.euron euro.2015.08.003
Toriumi, K., Tanaka , J., Mamiya, T., A lkam, T., Kim, H. C., Nitta, A., &
Nabeshima, T. (2018). Shati/Nat8l knockout mice show behav-
ioral def icits ameliorated by atomoxetine and methylphenidate.
Behavioral Brain Research, 339, 207–214. http s://doi.org /10.1016/ j.
bbr.2017.11.040
Uchida, S., Hara, K., Kobayashi, A., Fujimoto, M., Otsuki, K., Yamagata,
H., Watanabe, Y. (2011). Impaired hippocampal spinogenesis and
neurogenesis and altered affec tive behavior in mice lacking heat
shock factor 1. Proceedings of the National Academy of Sciences of the
United States of America, 108, 1681–1686. https://doi.org/10.1073/
pnas.1016 4 24108
van Pelt, J., & van Ooyen, A. (2013). Estimating neuronal connectivity
from axonal and dendritic density fields. Frontiers in Computational
Neuroscience, 7, 160.
Wang, D., Noda , Y., Zhou, Y., Mouri, A ., Mizoguchi, H., Nit ta, A ., …
Nabeshima, T. (2007). The allosteric potentiation of nicotinic ace-
tylcholine receptors by galantamine ameliorates the cognitive dys-
function in beta amyloid25-35 i.c.v.-injected mice: Involvement of
dopaminergic systems. Neuropsychopharmacology, 32, 1261–1271.
https://doi.org/10.1038/sj.npp.1301256
Wang, Y., Jia, Y., Chen, X., Ling, X., Liu, S., Xu, G ., & Huang, L. (2012).
Hippocampal N-acetylaspartate and morning cortisol levels in
drug-naive, first-episode patients with major depressive disorder:
Effects of treatment. Journal of Psychopharmacology, 26, 146 3–1470.
https ://doi.or g/10.1177/02698 81112 450781
Wulaer, B., Nagai, T., Sobue, A., Itoh, N., Kuroda, K., Kaibuchi, K., …
Yamada, K. (2018). Repetitive and compulsive-like behaviors lead
to cognitive dysfunction in Disc1(Delta2-3/Delta2-3) mice. Genes,
Brain, and Behavior, 17, e12478.
Zhang, T.-Y., Keown, C. L., Wen, X., Li, J., Vousden, D. A., Anacker, C.,
Meaney, M. J. (2018). Environmental enrichment increases tran-
scriptional and epigenetic differentiation between mouse dorsal and
14 
|
   WULAER Et AL.
ventral dentate gyrus. Nature Communications, 9, 298. https://doi.
o r g / 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 1 7 - 0 2 7 4 8 - x
Zhang, W., Gu, G . J., Zhang, Q., Liu, J. H., Zhang, B., Guo, Y., … Xu, J. R.
(2017). NSCs promote hippocampal neurogenesis, metabolic changes
and synaptogenesis in APP/PS1 transgenic mice. Hippocampus, 27,
1250–1263. https://doi.org/10.1002/hipo.22794
SUPPORTING INFORMATION
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Supporting Information section.
How to cite this article: Wulaer B, Kunisawa K, Hada K, et al.
Shati/Nat8l deficiency disrupts adult neurogenesis and
causes at tentional impairment through dopaminergic
neuronal dysfunction in the dentate gyrus. J Neurochem.
2020;00:1–14. https://doi.org/10.1111/jnc.15022
... At this stage of knowledge about NAT8L, it is difficult to assume the exact role of such compartmentation, although this event corresponds with changes in WB results ( Figure 7A-D). The molecular NAT8L mass is estimated as 36 kDa [52], which remained insensitive to theophylline challenge ( Figure 7C2,D). However, the lysates developed with different anti-NAT8L antibodies showed extra Western blot bands occurring between 50 kDa and 100 kDa ( Figure 7C2), which might correspond with the Madhavarao and colleagues' findings, showing NAT8L involved in active biological complex with molecular mass about 670 kDa and at least 10 other bands indicating NAT8L complexes [53]. ...
... To reveal astrocytes and their immunoreactivity, brain homogenates were immunoassayed with antibodies against GFAP, EAAT1 and S100β ( Figure 6E-G). According to the protein levels of these markers, theophylline-triggered unstable NAA metabolism did not significantly affect astrogliosis, as it has been noted by other researchers [5,52,[55][56][57]. ...
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... Some progenitor cells are highly restricted to regions of the adult brain, such as the DG, which plays a pivotal role in ensuring lifelong neurogenesis in the mammalian brain, necessary for improved learning, memory, and overall cognitive abilities 45 . Indeed, changes in the production, growth, and overall regulation of new neurons in neurogenic brain regions have been associated with neuropsychiatric disorders, including ADHD 46,47 . Remarkably, retroviral THRSP overexpression reduced hippocampal neural stem progenitor cell proliferation when compared with that induced by THRSP knockdown, indicating that THRSP plays a role in NSC modulation essential for neurogenesis. ...
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... A role of NAAG in the latter is suggested by a recent study that identified GCPII mutation in humans that led to higher enzymatic activity and therefore reduced NAAG levels and is associated with deficits in working memory (Zink et al., 2020). Furthermore, Shati/Nat8l −/− mice are impaired in an object-based attention test (Wulaer et al., 2020), though it is unclear whether this results from deficiency in NAAG or its precursor NAA, which both are reduced in Nat8l −/− mice. Intact working memory in Rimkla −/− mice, as indicated by normal performance in the spontaneous alternating T-maze, does not exclude a role of NAAG in short-term memory but may indicate that Rimklb/NAAGS-I-dependent NAAG synthesis is required or can, in that case, compensate loss of NAAGS-II-dependent NAAG synthesis. ...
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... Since its establishment, the test has become a popular method to assess attention and related underlying mechanisms in different mouse models [6][7][8]. Among many tasks that have been developed to assess attentional functions in rodents, OBAT is easy to perform and does not require expensive equipment, and therefore may be accessible to investigators on a tight budget. ...
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Abstract Disturbances of attention are a common behavioral feature associated with neuropsychiatric disorders with largely unknown underlying causes. We previously developed an object-based attention test (OBAT) as a simple and practical method for evaluating attention in mice. Since its establishment, the test has become a popular method for assessing attention and related underlying mechanisms in various mouse models. However, the underlying neuronal network involved in this test has yet to be studied. The purpose of this study was to identify the principal brain regions activated in the OBAT. Accordingly, C57BL/6J mice were subjected to the OBAT and thereafter prepared for immunohistochemical quantification of c-Fos, an immediate early gene that is frequently used as a marker of neuronal activity, in 13 different brain regions. The number of c-Fos-positive cells was significantly higher in the prefrontal cortex (PFC), dorsomedial striatum (DMS), and dentate gyrus (DG) in the test group as compared to the control group. The neuronal activation of these brain regions during the OBAT indicates that these brain regions are necessary for the regulation of attention in this test. This was supported by excitotoxic lesioning of these brain regions, leading to impaired attention without causing locomotor dysfunction. This study is one of the first attempts to analyze the brain regions that regulate attention in the OBAT. These findings provide an initial insight into the role of these brain regions and ideas for studying the underlying neural and molecular mechanisms.
... No exclusion criteria were pre-determined, and no animals were excluded. Sample size for each experiment was determined based on our previous studies with the relevant type of experiment (Miwa et al., 2011;Mouri et al., 2018;Wulaer et al., 2020) ...
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Indoleamine 2,3-dioxygenase 1 (IDO1) is the first rate-limiting enzyme that metabolizes tryptophan to the kynurenine pathway. Its activity is highly inducible by pro-inflammatory cytokines and correlates with the severity of major depressive disorder (MDD). MicroRNAs (miRNAs) are involved in gene regulation and the development of neuropsychiatric disorders including MDD. However, the role of miRNAs in targeting IDO1 in the pathophysiology of MDD is still unknown. In the present study, we investigated the role of novel miRNAs in the regulation of IDO1 activity and its effect on lipopolysaccharide (LPS)-induced depression-like behavior in mice. LPS upregulated miR-874-3p concomitantly with increase of IDO1 expression in the prefrontal cortex (PFC), increase of immobility in the forced swimming test as depression-like behavior and decrease of locomotor activity as sickness behavior without motor dysfunction. The miR-874-3p increased in both neuron and microglia after LPS. Its mimic significantly suppressed LPS-induced IDO1 expression in the PFC. Infusion of IDO1 inhibitor (1-methyl-l-tryptophan) and miR-874-3p into PFC prevented an increase of immobility in the forced swimming test, but did not decrease of locomotor activity induced by LPS. These results suggest that miR-874-3p may play an important role in preventing the LPS-induced depression-like behavior through inhibition of IDO1 expression. This may also serve as a novel potential target molecule for the treatment of MDD.
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Schizophrenia (SCZ) is a severe psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits. Although its pathoetiology remains unclear, it is known to involve small GTPase signaling. Rho kinase, an effector of small GTPase Rho, is highly expressed in the brain and plays a major role in neurite elongation and neuronal architecture. This study used a touchscreen-based visual discrimination (VD) task to investigate the effects of Rho kinase inhibitors on cognitive impairment in a methamphetamine (METH)–treated mouse model of SCZ. Systemic injection of the Rho kinase inhibitor fasudil dose-dependently ameliorated METH-induced impairment of VD. Fasudil also significantly suppressed the increase in the number of c-Fos–positive cells in the medial prefrontal cortex (mPFC) and dorsomedial striatum (DMS) following METH treatment. Bilateral microinjections of Y-27632, the other Rho kinase inhibitor, into the mPFC or DMS significantly ameliorated the METH-induced impairment of VD. Two proteins downstream of Rho kinase, myosin phosphatase-targeting subunit 1 (MYPT1; Thr696) and myosin light chain kinase 2 (MLC2; Thr18/Ser19), exhibited increased phosphorylation in the mPFC and DMS, respectively, after METH treatment, and fasudil inhibited these increases. Both haloperidol and fasudil ameliorated the METH-induced impairment of VD, while clozapine had little effect. Both haloperidol and clozapine suppressed METH-induced hyperactivity, but fasudil had no effect. Finally, haloperidol and clozapine significantly suppressed the METH-induced increase in MYPT1 (Thr696) phosphorylation in the mPFC, but not in the DMS. Thus, the METH-induced cognitive function impairment was ameliorated by treatment with Rho kinase inhibitors, which may act via the cortico-striatal circuit.
Article
A novel N-acetyltransferase, Shati/Nat8l, was identified in the brains of mice exposed to methamphetamine. Shati/Nat8l overexpression in the medial prefrontal cortex (mPFC) was found to attenuate methamphetamine-induced dependence. The mPFC is a brain region that plays an important role in cognitive function. However, the effect of Shati/Nat8l on cognition and memory has not yet been clarified. To understand the role of Shati/Nat8l in memory, we generated C57BL/6J mice with overexpressed Shati/Nat8l in the mPFC and performed memory-related experiments, including novel object-location and object-in-context tests. Furthermore, we used quantitative immunohistochemistry to assess the presynaptic and postsynaptic proteins, synaptophysin and postsynaptic density protein (PSD)-95, respectively. Shati/Nat8l overexpression in the mPFC impaired both novel object-location and object-in-context memory. Moreover, Shati/Nat8l overexpression in the mPFC reduced PSD-95 levels, but not synaptophysin levels in the mPFC. These results demonstrated that Shati/Nat8l overexpression in the mPFC is involved in location and contextual memory, and can affect the excitatory postsynaptic protein, PSD-95.
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Prenatal nicotine exposure (PNE) causes behavioral abnormalities in offspring, such as an enhancement of impulsivity and decrease in attention at adolescence. Here we examined the effects of galantamine (GAL) on the behavioral and electrophysiological changes induced by PNE in mice. Pregnant C57BL/6J mice were exposed to nicotine (0.2 mg/mL) dissolved in sweetened (2% saccharin) drinking water during gestational day 14 and perinatal day 0 (P0). At the ages of postnatal days 42-49 (P42-P49), female offspring displayed impulsivity in the cliff avoidance test and impairment of visual attention in the object-based attention test. Decrease of long-term potentiation (LTP) and extracellular glutamate levels were observed in the prefrontal cortex of PNE mice. Systemic treatment with GAL (1 mg/kg, s.c.), an allosteric potentiating ligand for the nicotinic acetylcholine receptor (nAChR) and a weak cholinesterase inhibitor, attenuated the enhancement of impulsivity and impairment of attention induced by PNE in mice. Further, GAL reversed the impairment of LTP induced by PNE in the prefrontal cortex of mice, although it failed to attenuate the decrease of extracellular glutamate levels. The effects of GAL were blocked by an α 7 nAChR antagonist, methyllycaconitine (1 mg/kg, i.p.). These results suggest that PNE during cortex development affects nicotinic cholinergic-dependent plasticity and formation of impulsivity and attention. Furthermore, GAL could be a useful drug for cognitive impairments-related to attention deficit hyperactivity disorder.
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Chronic stress, a suggested precipitant of brain pathologies, such as depression and Alzheimer’s disease, is known to impact on brain plasticity by causing neuronal remodeling as well as neurogenesis suppression in the adult hippocampus. Although many studies show that stressful conditions reduce the number of newborn neurons in the adult dentate gyrus (DG), little is known about whether and how stress impacts on dendritic development and structural maturation of these newborn neurons. We, herein, demonstrate that chronic stress impacts differentially on doublecortin (DCX)-positive immature neurons in distinct phases of maturation. Specifically, the density of the DCX-positive immature neurons whose dendritic tree reaches the inner molecular layer (IML) of DG is reduced in stressed animals, whereas their dendritic complexity is increased. On the contrary, no change on the density of DCX-positive neurons whose dendritic tree extends to the medial/outer molecular layer (M/OML) of the DG is found under stress conditions, whereas the dendritic complexity of these cells is diminished. In addition, DCX+ cells displayed a more complex and longer arbor in the dendritic compartments located in the granular cell layer of the DG under stress conditions; on the contrary, their dendritic segments localized into the M/OML were shorter and less complex. These findings suggest that the neuroplastic effects of chronic stress on dendritic maturation and complexity of DCX+ immature neurons vary based on the different maturation stage of DCX-positive cells and the different DG sublayer, highlighting the complex and dynamic stress-driven neuroplasticity of immature neurons in the adult hippocampus.
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Shati/Nat8l is a novel N‐acetyltransferase identified in the brain of mice treated with methamphetamine (METH). Shati/Nat8l mRNA is expressed in various brain areas, including the prefrontal cortex (PFC), where the expression level is higher than that in other brain regions. Shati/Nat8l overexpression in the nucleus accumbens (NAc) attenuates the pharmacological response to METH via mGluR3. Meanwhile, dopamine (DA) and glutamate dysregulations have been reported in the medial prefrontal cortex (mPFC) and NAc after METH self‐administration and during reinstatement. However, the mechanism, the reward system, and function of Shati/Nat8l in the mPFC is unclear. Here, we injected an adeno‐associated virus (AAV) vector containing Shati/Nat8l into the mPFC of mice, to overexpress Shati/Nat8l in the mPFC (mPFC‐Shati/Nat8l). Interestingly, the METH‐induced conditioned place preference (CPP) was attenuated in the mPFC‐Shati/Nat8l mice, but locomotor activity was not. Additionally, immunohistochemical results from mice that were injected with AAV‐GFP showed fluorescence in the mPFC and other brain regions, mainly the NAc, indicating an mPFC‐NAc top‐down connection. Finally, in vivo microdialysis experiments revealed that Shati/Nat8l overexpression in the mPFC reduced extracellular DA levels and suppressed the METH‐induced DA increase in the NAc. Moreover, decreased extracellular glutamate levels were observed in the NAc. These results indicate that Shati/Nat8l overexpression in the mPFC attenuates METH‐induced CPP by decreasing extracellular DA in the NAc. In contrast, Shati/Nat8l‐mPFC overexpression did not alter METH‐induced hyperlocomotion. This study demonstrates that Shati/Nat8l in the mPFC attenuates METH reward‐seeking behaviour but not the psychomotor activity of METH.
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Early life experience influences stress reactivity and mental health through effects on cognitive-emotional functions that are, in part, linked to gene expression in the dorsal and ventral hippocampus. The hippocampal dentate gyrus (DG) is a major site for experience-dependent plasticity associated with sustained transcriptional alterations, potentially mediated by epigenetic modifications. Here, we report comprehensive DNA methylome, hydroxymethylome and transcriptome data sets from mouse dorsal and ventral DG. We find genome-wide transcriptional and methylation differences between dorsal and ventral DG, including at key developmental transcriptional factors. Peripubertal environmental enrichment increases hippocampal volume and enhances dorsal DG-specific differences in gene expression. Enrichment also enhances dorsal-ventral differences in DNA methylation, including at binding sites of the transcription factor NeuroD1, a regulator of adult neurogenesis. These results indicate a dorsal-ventral asymmetry in transcription and methylation that parallels well-known functional and anatomical differences, and that may be enhanced by environmental enrichment.
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Several neuropsychiatric and neurodegenerative disorders share stress as a risk factor and are more prevalent in women than in men. Corticotropin-releasing factor (CRF) orchestrates the stress response, and excessive CRF is thought to contribute to the pathophysiology of these diseases. We previously found that the CRF1 receptor (CRF1) is sex biased whereby coupling to its GTP-binding protein, Gs, is greater in females, whereas β-arrestin-2 coupling is greater in males. This study used a phosphoproteomic approach in CRF-overexpressing (CRF-OE) mice to test the proof of principle that when CRF is in excess, sex-biased CRF1 coupling translates into divergent cell signaling that is expressed as different brain phosphoprotein profiles. Cortical phosphopeptides that distinguished female and male CRF-OE mice were overrepresented in unique pathways that were consistent with Gs-dependent signaling in females and β-arrestin-2 signaling in males. Notably, phosphopeptides that were more abundant in female CRF-OE mice were overrepresented in an Alzheimer's disease (AD) pathway. Phosphoproteomic results were validated by demonstrating that CRF overexpression in females was associated with increased tau phosphorylation and, in a mouse model of AD pathology, phosphorylation of β-secretase, the enzyme involved in the formation of amyloid β. These females exhibited increased formation of amyloid β plaques and cognitive impairments relative to males. Collectively, the findings are consistent with a mechanism whereby the excess CRF that characterizes stress-related diseases initiates distinct cellular processes in male and female brains, as a result of sex-biased CRF1 signaling. Promotion of AD-related signaling pathways through this mechanism may contribute to female vulnerability to AD.Molecular Psychiatry advance online publication, 18 October 2016; doi:10.1038/mp.2016.185.
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Tremendous efforts have been spent on the development of electrical energy storage (EES) systems with high volumetric performance in the past few years due to the evergrowing demand of miniaturized, portable electronic devices, and electric vehicles. Among all the EES devices, supercapacitors with electrode materials derived from biosources have attracted special attention due to their eco‐friendliness, natural abundance, their intrinsic porous structures as well as their renewable and sustainable features. However, the relatively low packing densities make their specific volumetric capacitance intrinsically low, which has largely limited their further application in the supercapacitors. To address these issues, various promising approaches ranging from structural manufacture to compositional design are applied and significant breakthroughs are witnessed in recent years. In this progress report, key factors influencing the volumetric performance of biomass‐derived electrode materials are systematically discussed with a particular focus spanning from fundamental to operational aspects. This work provides insights into the development of high‐volumetric‐performance biomass‐derived supercapacitors by comprehensively summarizing recent advances in the rational structural design and fabrication. Perspectives regarding the future challenges and promising research directions on the design of next‐generation EES devices are also provided.
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Disrupted‐in‐schizophrenia 1 (Disc1) is a key molecular driver for the biology of mental diseases. In order to investigate its role in brain function, we previously generated mice lacking exons 2 and 3 of Disc1 on a C57BL/6J genetic background (Disc1Δ2‐3/Δ2‐3 mice), which have a deficiency of the full‐length Disc1 protein. In the present study, we examined the role of Disc1 in cognitive function using a touchscreen‐based visual discrimination task in which mice had to discriminate one of two stimuli simultaneously displayed on the screen and received a liquid reward. Disc1Δ2‐3/Δ2‐3 mice showed impaired performance in the VD task, and this was mainly attributed to the perseverative response being significantly stronger than that in wild‐type (WT) mice. Furthermore, the numbers of marbles buried in the marble burying test and nestlets shredded in the nestlet shredding test by Disc1Δ2‐3/Δ2‐3 mice were significantly higher than those by WT mice, suggesting perseverative/compulsive behaviors by Disc1Δ2‐3/Δ2‐3 mice. A treatment with clozapine ameliorated behavioral deficits in the VD and marble burying tasks. c‐Fos expression was significantly stronger in the dorsomedial striatum (DMS), but not the dorsolateral striatum (DLS) after the first VD session in Disc1Δ2‐3/Δ2‐3 mice than in WT mice. The treatment of mice that had previously expressed hM3Dq in the DMS with clozapine‐N‐oxide (CNO) impaired performance in the VD task. These results suggest that cognitive impairments accompanied by perseverative/compulsive behaviors in Disc1Δ2‐3/Δ2‐3 mice are associated with hyperactivity of the DMS.
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In the central nervous system, major histocompatibility complex class I (MHCI) molecules are mainly expressed in neurons, and neuronal MHCI have roles in synapse elimination and plasticity. However, the pathophysiological significance of astroglial MHCI remains unclear. We herein demonstrate that MHCI expression is up-regulated in astrocytes in the medial prefrontal cortex (mPFC) following systemic immune activation by an intraperitoneal injection of polyinosinic-polycytidylic acid (polyI:C) or hydrodynamic interferon (IFN)-γ gene delivery in male C57/BL6J mice. In cultured astrocytes, MHCI/H-2D largely co-localized with exosomes. To investigate the role of astroglial MHCI, H-2D, or sH-2D was expressed in the mPFC of male C57/BL6J mice using an adeno-associated virus vector under the control of a glial fibrillary acidic protein promoter. The expression of astroglial MHCI in the mPFC impaired sociability and recognition memory in mice. Regarding neuropathological changes, MHCI expression in astrocytes significantly activated microglial cells, decreased parvalbumin-positive cell numbers, and reduced dendritic spine density in the mPFC. A treatment with GW4869 that impairs exosome synthesis ameliorated these behavioral and neuropathological changes. These results suggest that the overexpression of MHCI in astrocytes affects microglial proliferation as well as neuronal numbers and spine densities, thereby leading to social and cognitive deficits in mice, possibly via exosomes created by astrocytes.
Article
We previously identified a novel molecule, SHATI/NAT8L, as having an inhibitory effect on methamphetamine dependence. We generated Shati/Nat8l knockout (KO) mice and found that they showed neurochemical changes and behavioral abnormalities related to attention deficit/hyperactivity disorder (AD/HD). In this study, we assessed validities of the Shati/Nat8l KO mice as a new animal model for AD/HD through a behavioral pharmacology approach. We conducted a locomotor activity test in a novel environment, a cliff avoidance test, and an object-based attention assay using Shati/Nat8l KO mice at the ages of 4 and 8 weeks. We found that at the ages of both 4 and 8 weeks, Shati/Nat8l KO mice showed hyperactivity in locomotor activity test, shortened jumping latency in cliff avoidance test, and lower recognition index in object-based recognition test. Moreover, we evaluated the effects of atomoxetine (ATX) and methylphenidate (MPH) on the behavioral deficits in Shati/Nat8l KO mice. As the result, almost all behavioral deficits were improved by the treatment of both ATX and MPH. Our findings suggest that Shati/Nat8l KO mice have an impaired neural system similar to AD/HD pathophysiology. Shati/Nat8l KO mice might serve as a novel and a useful animal model for the pathophysiology of AD/HD.
Article
Adult neurogenesis and synaptic remodeling persist as a unique form of structural and functional plasticity in the hippocampal dentate gyrus (DG) and subventricular zone (SVZ) of the lateral ventricles due to the existence of neural stem cells (NSCs). Transplantation of NSCs may represent a promising approach for the recovery of neural circuits. Here, we aimed to examine effects of highly neuronal differentiation of NSCs transplantation on hippocampal neurogenesis, metabolic changes and synaptic formation in APP/PS1 mice. 12-month-old APP/PS1 mice were used for behavioral tests, immunohistochemistry, western blot, transmission electron microscopy and proton magnetic resonance spectroscopy (1H-MRS). The results showed that N-acetylaspartate (NAA) and Glutamate (Glu) levels were increased in the Tg-NSC mice compared with the Tg-PBS and Tg-AD mice 10 weeks after NSCs transplantation. NSC-induced an increase in expression of synaptophysin and postsynaptic protein-95, and the number of neurons with normal synapses was significantly increased in Tg-NSC mice. More doublecortin-, BrdU/NeuN- and Nestin-positive neurons were observed in the hippocampal DG and SVZ of the Tg-NSC mice. This is the first demonstration that engrafted NSCs with a high differentiation rate to neurons can enhance neurogenesis in a mouse model of AD and can be detected by 1H-MRS in vivo. It is suggested that engraft of NSCs can restore memory and promote endogenous neurogenesis and synaptic remodeling, moreover, 1H-MRS can detect metabolite changes in AD mice in vivo. The observed changes in NAA/creatine (Cr) and glutamate (Glu)/Cr may be correlated with newborn neurons and new synapse formation. This article is protected by copyright. All rights reserved.
Article
Stress, a well-known sculptor of brain plasticity, is shown to suppress hippocampal neurogenesis in the adult brain; yet, the underlying cellular mechanisms are poorly investigated. Previous studies have shown that chronic stress triggers hyperphosphorylation and accumulation of the cytoskeletal protein Tau, a process that may impair the cytoskeleton-regulating role(s) of this protein with impact on neuronal function. Here, we analyzed the role of Tau on stress-driven suppression of neurogenesis in the adult dentate gyrus (DG) using animals lacking Tau (Tau-knockout; Tau-KO) and wild-type (WT) littermates. Unlike WTs, Tau-KO animals exposed to chronic stress did not exhibit reduction in DG proliferating cells, neuroblasts and newborn neurons; however, newborn astrocytes were similarly decreased in both Tau-KO and WT mice. In addition, chronic stress reduced phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)/glycogen synthase kinase-3β (GSK3β)/β-catenin signaling, known to regulate cell survival and proliferation, in the DG of WT, but not Tau-KO, animals. These data establish Tau as a critical regulator of the cellular cascades underlying stress deficits on hippocampal neurogenesis in the adult brain.Molecular Psychiatry advance online publication, 30 May 2017; doi:10.1038/mp.2017.103.