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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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WULAER Et AL.
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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WULAER Et AL.
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 2k–l) 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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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
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
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
