![loss and/or the development of cognitive deficits that are
associated with certain neuropathological conditions.
Thus, neurogenic drugs present potential therapeutic
value for treatment of these disorders (Taupin, 2011).
Screening analysis of chemical libraries in stem cell-
based assays has identified several pro-neurogenic
small molecules with therapeutic potential (Schneider
et al., 2008; Pieper et al., 2010; Wurdak et al., 2010).
Within this context, isoxazole 9 [Isx-9; N-cyclopropyl-5-(t
hiophen-2-yl)isoxazole-3-carboxamide] was reported to
influence stem-cell fate both in vitro and in vivo, namely
by inducing a robust increase in neuronal differentiation.
This effect seems to occur through the modulation of myo-
cyte enhancer factor-2 (Mef2) (Schneider et al., 2008;
Petrik et al., 2012), a family of transcription factors that
plays a key role in the activation of genetic programs that
control cell differentiation, proliferation, morphogenesis,
survival and apoptosis (Potthoff and Olson, 2007).
In a previous in vivo study, it was demonstrated that
Isx-9 crosses the blood–brain barrier and is a safe
pharmacological approach to increase neurogenesis in
the SGZ of the hippocampal DG in adult mice (Petrik
et al., 2012). However, despite being a promising syn-
thetic neurogenic compound, our current knowledge on
its in vivo properties is still limited. Thus, in the present
study we confirmed and expanded the results initially
reported by Petrik et al. (2012) and demonstrated the
neurogenic effects of Isx-9 on cell proliferation and neu-
ronal commitment in the adult rat hippocampal DG follow-
ing repeated exposure to a commonly used laboratory
procedure that is potentially associated with increased
levels of stress (repeated intraperitoneal, i.p., injections).
We found that Isx-9 was able to reverse the reduction in
hippocampal cell proliferation and neuronal commitment
found in vehicle-treated animals, further highlighting the
neurogenic properties of this compound. Thus, the devel-
opment of synthetic molecules structurally and function-
ally related to Isx-9 that possess a greater half-life than
this compound (thus reducing the frequency of adminis-
tration) may prove to have therapeutic value for the treat-
ment of conditions associated with an increase in stress
levels.
EXPERIMENTAL PROCEDURES
Synthesis of Isx-9
Isx-9 was prepared according to the method of Schneider
et al. with minor modifications (Fig. 1). All reactions were
performed in oven- or flame-dried glassware, under a
positive pressure of argon, unless otherwise indicated.
Organic solutions were concentrated between 35 and
40 °C by rotary evaporation under vacuum. All reagents
were used as received from commercial suppliers
unless otherwise indicated. Commercial solvents were
used as received with the following exceptions.
Dichloromethane, tetrahydrofuran and toluene were
dried by passage through a column of alumina in a
commercial solvent purification system. Methanol was
dried over activated 4 A˚ MS. 1
H chemical shifts are
reported in parts per million (ppm, d scale) downfield
from tetramethylsilane, and are referenced to residual
protium in the NMR solvent (CDCl3: d 7.26; (CD3)2CO: d
2.05). Likewise, 13
C chemical shifts are referenced to
the carbon resonances of the solvent (CDCl3: d 77.16;
(CD3)2CO: d 29.85). Infrared spectra were collected
using an FT-IR spectrometer.
(Z)-Methyl 4-hydroxy-2-oxo-4-(thiophen-2-yl)but-3-en
oate (1). To a stirred solution of 2-acetylthiophene
(2.57 mL, 23.8 mmol) and dimethyloxylate (3.72 g,
31.5 mmol) in 125 mL dry toluene was added 30 mL of a
solution of t-BuOK (1.0 M in THF, 30.0 mmol) dropwise
at room temperature. Once addition was complete, the
resultant slurry was stirred overnight at room
temperature ($16 h). The reaction was quenched with
60 mL of 1 N HCl (aq) and the two phases were
separated. The organic layer was washed with water and
then brine, dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The crude product was
recrystallized from hexanes to afford compound 1 as a
bright yellow powder in 86% yield. 1
H NMR (300 MHz,
CDCl3): d 7.85 (dd, J = 3.8, 1.2 Hz, 1H), 7.72 (dd,
J = 5.0, 1.2 Hz, 1H), 7.19 (dd, J = 5.0, 3.8 Hz, 1H), 6.93
(s, 1H), 3.94 (s, 3H); 13
C NMR (75 MHz, CDCl3) d 186.2
(C), 164.6 (C), 162.7 (C), 142.2 (C), 135.4 (CH), 132.8
(CH), 128.9 (CH), 99.8 (CH), 53.3 (CH3); IR (KBr, cmÀ1
)
3114 (m), 3092 (m), 3076 (w), 2964 (m), 1751 (m), 1738
(s), 1611 (s), 1426 (s), 1250 (s), 1228 (s), 1121 (s), 1063
(m), 968 (m), 826 (m), 776 (s), 732 (s), 682 (m).
Methyl 5-(thiophen-2-yl)isoxazole-3-carboxylate (2).
N-Hydroxylamine hydrochloride (1.67 g, 24.0 mmol) was
added to a stirring solution of ketone 1 (4.21 g,
19.8 mmol) in 100 mL of dry methanol and the reaction
mixture was then heated to 60 °C for 12 h. The resultant
solution was then concentrated under reduced pressure
and the precipitated beige solid was resuspended in
100 mL of water. The resulting slurry was stirred at
room temperature for 1 h before the product was
collected by filtration through a sintered glass fritted
funnel to yield compound 2 as a beige solid in
quantitative yield. 1
H NMR (300 MHz, CDCl3): d 7.56
(dd, J = 3.8, 1.2 Hz, 1H), 7.50 (dd, J = 5.0, 1.2 Hz,
1H), 7.14 (dd, J = 5.1, 3.7 Hz, 1H), 6.78 (s, 1H), 3.99
(s, 3H); 13
C NMR (75 MHz, CDCl3) d 167.0 (C), 160.4
(C), 156.8 (C), 129.0 (CH), 128.4 (CH), 128.3 (C), 128.0
(CH), 99.7 (CH), 53.1 (CH3); IR (KBr, cmÀ1
) 3134 (s),
3116 (m), 3083 (w), 2954 (m), 1731 (s), 1593 (s), 1476
(s), 1452 (s), 1275 (s), 1250 (s), 1138 (s), 1004 (s), 930
(s), 853 (s), 810 (s), 779 (s), 703 (s).
5-(Thiophen-2-yl)isoxazole-3-carboxylic acid (3). An
aqueous solution of LiOH (1.0 M, 140 mL) was added to
a stirred solution of ester 2 (16.9 g, 80.6 mmol) in 90 mL
of THF and reacted at 50 °C for 3 h. The reaction
mixture was cooled to room temperature and the
aqueous layer was acidified to pH 1 (with 3 M HCl) and
then extracted with ethyl acetate (2 Â 100 mL). The
combined organic extracts were washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated
in vacuo to afford compound 3 as a yellow crystalline
solid in 71% yield. 1
H NMR (300 MHz, (CD3)2CO): d
7.79 (dd, J = 5.1, 1.0 Hz, 1H), 7.76 (dd, J = 3.5,
1.2 Hz, 1H), 7.26 (dd, J = 5.1, 3.7 Hz, 1H), 7.06 (s, 1H);
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![vibratome (Leica VT1000S, Nussloch, Germany) at 30-
lm thickness. Sections were collected in a 1/6-section-
sampling fraction and stored in a cryoprotectant solution
[0.04 M Tris-buffered saline (TBS), 30% ethylene
glycerol, 30% glycerol] at 4 °C.
Immunohistochemistry
One series of free-floating brain sections was processed
for BrdU immunohistochemistry as previously described
(Gil-Mohapel et al., 2011). Briefly, after thorough rinsing
in 0.1 M TBS buffer (84 mM Tris–HCl, 16 mM Tris, 0.9%
NaCl, pH = 7.4), the sections were incubated in 2 N
HCl at 65 °C for 30 min to denature the DNA. The sec-
tions were then pre-incubated for 1 h at room temperature
(24 °C) in 5% normal horse serum (NHS) and 0.25% Tri-
ton X-100 in 0.1 M TBS and then incubated for 48 h at
4 °C with a mouse monoclonal antibody against BrdU
(1:60, M0744; Dako, Glostrup, Denmark) in TBS contain-
ing 5% NHS. After incubation with a biotinylated horse
anti-mouse IgG secondary antibody (1:200, BA-2001;
Vector Laboratories, Burlingame, CA, USA) for 2 h at
room temperature, the bound antibodies were visualized
using an avidin-biotin-peroxidase complex system (Vec-
tastain ABC Elite kit PK4000; Vector Laboratories) with
2,2-diaminobenzidine (DAB, DAB kit SK 4100; Vector
Laboratories) as the chromogen. The sections were
mounted onto 2% gelatin-coated microscope slides,
dehydrated in a series of ethanol solutions of increasing
concentrations followed by a 5-min incubation with a
xylene substitute (CitriSolv; Fisher Scientific, Pittsburgh,
PA, USA), and coverslipped with Permount mounting
medium (Fisher Scientific).
An adjacent series of brain sections was also
processed for detection of the endogenous proliferative
marker Ki-67, a nuclear protein that is expressed during
all active phases of the cell cycle, but is absent from
cells at rest (Scholzen and Gerdes, 2000), as previously
described (Gil-Mohapel et al., 2011). Briefly, after thor-
ough rinsing, the sections were incubated in 10 mM
sodium citrate buffer (in 0.1 M TBS, pH = 6.0) at 95 °C
for 5 min. This step was repeated twice to completely
unmask the antigens. After quenching with 3%
H2O2/10% methanol in 0.1 M TBS for 15 min and pre-
incubating with 5% normal goat serum (NGS) for 1 h at
room temperature, the sections were incubated for 48 h
at 4 °C with a rabbit polyclonal primary antibody against
Ki-67 (1:500, VP-K451; Vector Laboratories). After thor-
ough rinsing, the sections were incubated for 2 h with
the secondary antibody (biotin-conjugated goat anti-
rabbit IgG, 1:200, BA-1000; Vector Laboratories) in 5%
blocking solution at room temperature. The bound anti-
bodies were detected using the avidin-biotin-peroxidase
complex system (Vector Laboratories) with DAB (Vector
Laboratories) as the chromogen. Brain sections were
mounted onto microscope slides as described for BrdU
immunolabelling (see details above).
Finally, an additional series of brain sections was
processed for NeuroD, a basic helix-loop-helix
transcription factor involved in early neuronal maturation
(Brunet and Ghysen, 1999; Miyata et al., 1999), as previ-
ously described (Gil-Mohapel et al., 2011). Briefly, after
quenching and pre-incubation with NHS at room temper-
ature, the sections were incubated for 48 h at 4 °C with
a goat anti-NeuroD primary antibody (1:200, SC-1084;
Santa Cruz Biotechnology, Santa Cruz, CA, USA). The
sections were then incubated for 2 h with the secondary
antibody (biotin-conjugated horse anti-goat IgG, 1:200,
BA-9500; Vector Laboratories) in 5% blocking solution
at room temperature. The bound antibodies were
detected using the avidin-biotin-peroxidase complex sys-
tem (Vector Laboratories) with DAB (Vector Laboratories)
as the chromogen. Brain sections were mounted onto
microscope slides as described for BrdU immunolabelling
(see details above).
Morphological quantification by conventional
microscopy
All morphological analyses were performed on coded
slides with the experimenter blinded to the identity of the
brain sections (i.e., the animal), using an Olympus BX51
microscope equipped with 10Â, 40Â, and 100Â
objectives (Olympus, Center Valley, PA, USA). Image
Pro-Plus software (version 6.0 for WindowsTM, Media
Cybernetic Inc., Silver Spring, MD, USA) and a Cool
Snap HQ camera (Photometrics, Tucson, AZ, USA)
were used for image capture. The total number of
labeled cells present in all sections obtained from a
single brain (i.e., animal) and containing the DG
hippocampal sub-region were calculated, and that total
number of cells were then used as the single data point
from that respective animal. For each animal (i.e.,
brain), calculations were performed as follows: the total
number of BrdU-, Ki-67- or NeuroD-immunopositive
cells present in the SGZ of either the entire DG (from
Bregma À2.30 to À6.04; approximately 20 coronal
sections per brain), the dorsal DG (from Bregma À2.30
to À4.16; approximately 10 coronal sections per brain),
or the ventral DG (Bregma À4.16 to À6.04;
approximately 10 coronal sections per brain) sub-
regions (Paxinos and Watson, 1986) were quantified by
manually counting all DAB-positive cells present with 2–
3 cell diameters of the SGZ. Results were expressed as
the total number of labeled cells in the DG hippocampal
sub-region of each individual brain (i.e., animal) by multi-
plying the average number of labeled cells/DG section by
the total number of 30-lm-thick sections obtained from
that respective animal and containing either the entire
DG (125 slices), the dorsal DG (62 slices), or the ventral
DG (63 slices). A single data point was entered for each
individual animal and group averages were calculated
as follows:
P
of total number of cells per brain (i.e., per
animal)/total number of brains (i.e., animals). Images
were processed with Adobe Photoshop 4.0 (Adobe Sys-
tems, Mountain View, CA, USA). Only contrast enhance-
ments and color level adjustments were made.
CORT assay
Blood tail samples were taken on the 14th day of
treatment to assess CORT levels in the animals.
Samples were stored at 4 °C overnight and then
centrifuged for 30 min at 3000g to collect the serum
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![(which was stored at À20 °C until processing). CORT
levels were determined using an enzyme immunoassay
kit (900-097, Assay Designs, Ann Habor, MI, USA)
following the manufacturer’s instructions. Samples were
run in duplicates. Briefly, the provided donkey anti-
sheep IgG-coated 96-well plate was loaded with a
CORT standard (in the range of 0–20,000 pg/ml) and
the serum samples. An alkaline phosphatase
conjugated to CORT and the polyclonal antibody against
CORT was added to the wells and the plate was then
gently shaken for 2 h at room temperature. Following
three washes, wells were aspirated and p-nitrophenyl
phosphate substrate solution was added and incubated
for 1 h at room temperature to start the reaction of the
alkaline phosphatase. The reaction was stopped by
adding stop solution containing trisodium phosphate and
CORT levels were determined at 405 nm with a
VersaMax microplate reader (Molecular Devices,
Sunnyvale, CA, USA) and analyzed with the SoftMax
Pro 5.2 software (Molecular Devices). CORT levels
were calculated from the standard curve prepared for
each plate and were expressed as ng/ml serum.
Statistical analysis
Statistical analyses were performed using the Statistica
7.1 analytical software (StatSoft Inc., Tulsa, OK, USA).
Sample distributions presented equal variances and
normal distributions (data not shown). Differences
among experimental groups were compared using two-
tailed unpaired Student’s t-tests or one-way analysis of
variance (ANOVA) followed by Tukey’s multiple range
post-hoc test when appropriate and correcting for
multiple comparisons when necessary. A p value of less
than 0.05 was considered to be statistically significant.
RESULTS
Effect of Isx-9 on hippocampal cell proliferation
To evaluate the effect of Isx-9 on adult hippocampal cell
proliferation, adult male Sprague–Dawley rats were
treated with this compound (20 mg/kg/day; i.p.) for
14 days. Animals received two doses of the exogenous
cell proliferation marker BrdU (150 mg/kg/i.p. injection;
12 h apart) on the 15th day and were sacrificed 12 h
after the last injection. Their brains were then processed
for immunohistochemistry against BrdU and
endogenous markers of cell proliferation and neuronal
commitment. Immunohistochemistry against BrdU
revealed a significant increase in the number of BrdU+
cells in Isx-9-treated animals in comparison with vehicle-
treated animals in the entire [t(12) = À4.81,
p = 0.0007, Student’s t-test], dorsal [t(12) = À3.072
p = 0.011, Student’s t-test] and ventral [t(12) = À4.33
p = 0.001, Student’s t-test] hippocampal DG (Fig. 2).
Since this exogenous cell proliferation marker is only
incorporated into the DNA of dividing cells during the S-
phase of the cell cycle, the exclusive use of BrdU may
underestimate the number of cells undergoing cell
division at any given time (and potentially fail to detect
less robust differences in cell proliferation) (Eisch and
Mandyam, 2007). Therefore, to confirm the results
obtained with BrdU and further elucidate whether overall
changes in cell proliferation (i.e., not just restricted to
the number of cells in S-phase of the cell cycle) occurred
with Isx-9 treatment, we also performed immunohisto-
chemistry for the endogenous cell cycle protein Ki-67,
which is expressed during all active phases of the cell
cycle (Christie and Cameron, 2006). One-way ANOVA
confirmed the proliferative effect of Isx-9 with a main
effect of treatment observed in the entire hippocampal
DG [F(2,21) = 8.87, p = 0.002]. Further post-hoc analysis
revealed a reduction in cell proliferation in rats that
received i.p. injections of vehicle during 14 days when
compared with naı¨ve animals (which were not manipu-
lated and did not receive any treatment during that time
period) (p = 0.003, Tukey’s post-hoc test). Furthermore,
this reduction in cell proliferation was prevented by Isx-9
treatment (p = 0.016, Tukey’s post-hoc test). Similar
results were also found both in the dorsal and ventral
aspects of the hippocampal DG [F(2,21) = 9.02,
p = 0.002 and F(2,21) = 5.21, p = 0.001, respectively],
where post-hoc analysis revealed that vehicle-injected
rats presented a significant reduction in Ki-67+ cells both
in the dorsal and ventral DG sub-regions (p = 0.004 and
p = 0.016, respectively, Tukey’s post-hoc test). This
effect was prevented by Isx-9 treatment in the dorsal
aspect of the hippocampal DG (p = 0.008, Tukey’s
post-hoc test) (Fig. 3).
Fig. 2. Effect of repeated Isx-9 administration (20 mg/kg, i.p.) on DG
cell proliferation as assessed by BrdU immunohistochemistry. A
significant increase in the number of BrdU-positive cells was
observed in rats treated for 14 days with Isx-9 in comparison with
vehicle-treated animals in the entire, dorsal and ventral aspects of the
hippocampal DG (A). Values are represented as mean ± SEM.
Numbers of rats per experimental group are indicated in the
respective bars. Representative photomicrographs of the effect of
repeated treatment with vehicle or Isx-9 on the number of cells that
incorporated the exogenous proliferation marker BrdU at 20Â (scale
bar = 50 lm) and 40Â (scale bar = 10 lm) magnification (B).
Examples of BrdU-positive cells are denoted by arrows. *
p < 0.05
and **
p < 0.01 in comparison with vehicle-treated group (Student’s t-
test).
216 L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222](https://image.slidesharecdn.com/90d156e9-5ee3-4761-848d-fd7048fb8cb2-160809181804/85/ISX9-Karthik-Gopalakrishnan-5-320.jpg)
![Effect of Isx-9 on the number of immature neurons in
the hippocampus
The effect of Isx-9 on the number of immature neurons in
the DG of the hippocampus was assessed by
immunostaining slices with the immature neuronal
marker NeuroD, as previously described by us (Boehme
et al., 2011; Gil-Mohapel et al., 2011, 2013; Kannangara
et al., 2014; Bettio et al., 2016). Statistical analysis
demonstrated a significant main effect of treatment in
the entire DG [F(2,20) = 8.12, p = 0.003]. Further post-
hoc analysis revealed a reduction in the number of Neu-
roD+ cells in the DG of rats treated with vehicle when
compared with naı¨ve animals (which were not manipu-
lated and did not receive any treatment during that time
period) (p = 0.002, Tukey’s post-hoc test). This decrease
in the number of immature neurons was prevented by
Isx-9 treatment (p = 0.025, Tukey’s post-hoc test).
Furthermore, evaluation of number of neuroblasts (i.e.,
expression of NeuroD) in the dorsal and ventral aspects
of the hippocampal DG also revealed a significant main
effect of treatment [F(2,20) = 5.36, p = 0.015 and
F(2,20) = 8.81, p = 0.002, respectively]. Post-hoc analy-
sis revealed that vehicle-treated rats showed a reduction
in the number of immature neurons in both dorsal and
ventral aspects of the hippocampal DG when compared
with naı¨ve animals (p = 0.018 and p = 0.001, respec-
tively, Tukey’s post-hoc test). Isx-9 treatment was able
to prevent this reduction both in the dorsal (p = 0.046,
Tukey’s post-hoc test) and ventral (p = 0.031, Tukey’s
post-hoc test) aspects of the hippocampal DG (Fig. 4).
Influence of Isx-9 on CORT levels
To evaluate the levels of stress experienced by the
animals as a consequence of the daily handling and
injections procedure (Balcombe et al., 2004; Titterness
and Christie, 2008), tail blood samples were obtained on
day 14 (i.e., the last day of vehicle- or Isx-9 treatment)
and circulating blood levels of CORT were measured. A
one-way ANOVA revealed a significant main effect of
Isx-9 treatment [F(2,21) = 4.13, p = 0.030]. Further post-
hoc analysis revealed a significant increase in the circulat-
ing levels of CORT in animals treated with Isx-9 when
compared with vehicle-treated rats (p = 0.035, Tukey’s
post-hoc test) (Fig. 5).
DISCUSSION
The present study confirmed the neurogenic properties of
Isx-9 previously observed in mice (Petrik et al., 2012) and
showed for the first time that repeated administration of
this compound (for 14 days) is able to significantly
increase stem cell proliferation and neuronal commitment
in the DG of the hippocampus of adult rats and reverse
the effects of repeated manipulation and injections on
these initial stages of the neurogenic process.
The hippocampus is a brain structure with a well-
established role in the modulation of learning and
memory (Whitlock et al., 2006; Bird and Burgess, 2008;
Neves et al., 2008) as well as in mood regulation
(Campbell and Macqueen, 2004; Ray et al., 2011). Since
this structure is one of the few brain regions where new
neurons are known to be born and to functionally integrate
Fig. 3. Effect of repeated Isx-9 administration (20 mg/kg, i.p.) on DG cell proliferation as assessed by Ki-67 immunohistochemistry. Cell proliferation
was significantly reduced in rats that received daily injections of vehicle [(2-hydroxypropyl)-b-cyclodextrin] for 14 days when compared to naı¨ve (i.e.,
non-manipulated) animals. This effect was reversed by Isx-9 treatment (20 mg/kg, i.p.) in the entire and dorsal aspect of the hippocampal DG (A).
Values are represented as mean ± SEM. Animal numbers for each condition are indicated within the respective bar in the graphs. Representative
photomicrographs of DG sections processed for Ki-67 immunohistochemistry from naı¨ve, vehicle- and Isx-9-treated rats at 20Â (scale bar = 50 lm)
and 40Â (scale bar = 10 lm) magnification (B). Examples of Ki-67-positive cells are denoted by arrows. *
p < 0.05 and **
p < 0.01 in comparison
with naı¨ve rats, #
p < 0.05 and ##
p < 0.01 in comparison with vehicle-treated animals.
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![into the pre-existing neuronal circuitry into adulthood, it is
now believed that adult hippocampal neurogenesis plays
a role in certain aspects of learning and memory as well
as mood disorders (Giovanello et al., 2004; Dranovsky
and Hen, 2006; Winocur et al., 2006; Becker and
Wojtowicz, 2007; Gil-Mohapel et al., 2013). Furthermore,
the hippocampus and its DG are not homogeneous struc-
tures, having different patterns of gene expression and
anatomical projections along their dorsal/ventral axis.
The dorsal hippocampus (defined as 50% of hippocampal
volume starting at the septal pole) communicates with
brain regions associated with cognition, and is therefore
primarily involved with processes of learning and memory.
On the other hand, the ventral hippocampus (defined as
50% of the hippocampal volume starting at the temporal
pole) is better situated to contribute to emotional
responses, having an important role in motivational and
affective behaviors (Bannerman et al., 2004; Fanselow
and Dong, 2010). In the present study, we did not observe
localized differences in the dorsal and ventral portions of
the hippocampal DG with regard to the reduction in cell
proliferation and number of neuroblasts induced by the
repeated injection procedure and the reversion of this def-
icit by Isx-9. This observation suggests that this com-
pound has a broad neurogenic action, being able to
potentiate both cell proliferation and neuronal commit-
ment in both anatomical aspects of this structure.
Although in our study we only used one marker of
neuronal commitment (NeuroD, an helix-loop-helix
transcription factor expressed in immature neurons or
neuroblasts; Brunet and Ghysen, 1999; Miyata et al.,
1999), the increase in cell proliferation induced by Isx-9
was mirrored by a similar increase in the number of
NeuroD+ cells. Therefore, it is likely that the majority of
proliferating cells observed in the hippocampal DG upon
Isx-9 treatment are committed to the neuronal lineage.
This conclusion is supported by the findings of
Schneider et al. (2008), which indicate that Isx-9, through
a mechanism involving N-methyl-D-aspartate (NMDA)
receptor-induced Ca2+
signaling and Mef2 transcription,
may indirectly activate NeuroD expression (Schneider
et al., 2008). In addition, the study by Petrik et al.
(2012) reinforces this conclusion by showing that Isx-9
leads to an increase in the proportion of BrdU/NeuN
double-labeled cells but not BrdU/glial fibrillary acid pro-
tein (GFAP) double-labeled cells (Petrik et al., 2012).
Interestingly, Schneider et al. (2008) also demonstrated
that Isx-9 not only induces robust neuronal differentiation,
but also significantly blocks competing astrocytic differen-
tiation in adult neural stem cells (Schneider et al., 2008).
This effect was also observed in a recent study where this
small synthetic molecule promoted the differentiation of
neural stem cells but negatively affected oligodendrocyte
precursor cells (OPCs) and endothelial progenitor cells
(EPCs) (Koh et al., 2015).
In our study, the Isx-9 solution was prepared using
HP-b-CD, a vehicle that presents low toxicity and is
commonly used for drug delivery to biological systems
(Gould and Scott, 2005). Surprisingly, animals that
received daily i.p. injections of vehicle during the 14-day
treatment period demonstrated reduced levels of DG cell
proliferation (as assessed with the cell proliferation mar-
Fig. 4. Effect of repeated Isx-9 administration (20 mg/kg, i.p.) on DG neuronal differentiation as assessed by NeuroD immunohistochemistry.
Animals injected with vehicle [(2-hydroxypropyl)-b-cyclodextrin] demonstrated a decrease in the number of NeuroD-positive neuroblasts in
comparison with naı¨ve rats in the entire, dorsal and ventral aspects of the hippocampal DG. This impairment in neuronal differentiation was reversed
by repeated Isx-9 administration in the entire DG (A). Values are represented as mean ± SEM. Numbers of animals per experimental group are
indicated within the respective bars. Representative photomicrographs of hippocampal DG of naı¨ve, vehicle- and Isx-9-treated rats on the
expression of the endogenous differentiation marker NeuroD at 20Â (scale bar = 50 lm) and 40Â (scale bar = 10 lm) magnification (B). Examples
of NeuroD-positive cells are denoted by arrows. *
p < 0.05 and **
p < 0.01 in comparison with naı¨ve rats, #
p < 0.05 and ##
p < 0.01 in comparison
with vehicle-treated animals.
218 L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222](https://image.slidesharecdn.com/90d156e9-5ee3-4761-848d-fd7048fb8cb2-160809181804/85/ISX9-Karthik-Gopalakrishnan-7-320.jpg)
![ker Ki-67; Fig. 2) and reduced number of neuroblasts (as
assessed with the immature neuronal marker NeuroD;
Fig. 3) when compared with animals that were not manip-
ulated and did not receive any i.p. injections during the
same period of time (i.e., naı¨ve rats). Given the low toxi-
city of the vehicle used (Gould and Scott, 2005), we
believe this decrease in hippocampal cell proliferation
and in the number of immature neurons may be a conse-
quence, at least in part, of the daily manipulations and
injections these animals received (i.e., procedure-
induced stress). In agreement, it is well-known that stress
negatively impacts hippocampal cell proliferation and
neuronal differentiation in mammals leading to a reduction
in hippocampal volume (Joe¨ ls et al., 2007; Lee et al.,
2009). The deleterious effects of stress on hippocampal
structural plasticity (i.e., adult neurogenesis) have been
repeatedly shown to occur in response to a large variety
of stressors including subordination stress (Gould et al.,
1997), resident-intruder stress (Gould et al., 1998), foot-
shock (Malberg and Duman, 2003; Vollmayr et al.,
2003), restraint stress (Pham et al., 2003; Bain et al.,
2004), isolation (Dong et al., 2004), cold immobilization
(Heine et al., 2004), cold swim (Lee et al., 2002; Heine
et al., 2004), and predator odor (Tanapat et al., 1998;
Falconer and Galea, 2003; Mirescu et al., 2004). Taking
this into account, it is not surprising that in our study,
14 days of daily manipulations and i.p. injections (which,
besides being a mildly painful procedure also involves
brief periods of restraint) might have been stressful
enough to cause a significant reduction in adult hip-
pocampal cell proliferation and neuronal survival and/or
differentiation.
On the other hand, despite the low toxicity of the
vehicle used to prepare the Isx-9 solution (HP-b-CD)
(Gould and Scott, 2005), some studies have raised con-
cerns regarding its safety since there is evidence for
hematological and histopathological alterations in rats
that received HP-b-CD through the intravenous route
(Gould and Scott, 2005; Kantner and Erben, 2012).
Regarding its effects on the central nervous system, var-
ious studies have shown that chronic administration of
HP-b-CD was not associated with neurotoxic effects when
administered either systemically (Yao et al., 2012; Matsuo
et al., 2013; Walenbergh et al., 2015) or through the
intracerebroventricular route (Aqul et al., 2011; Matsuo
et al., 2014). However, since the HP-b-CD-induced
histopathological changes observed in previous studies
(Gould and Scott, 2005; Kantner and Erben, 2012) were
found with HP-b-CD doses lower than those used in the
present study, we cannot rule out the possibility that
vehicle-induced neurotoxicity might underlie, at least in
part, the impairment on cell proliferation and/or neuronal
commitment observed in the present study in the
vehicle-treated group. In addition, the potential neurotox-
icity of HP-b-CD might have been overlooked in the study
by Petrik et al. (2012) (who also used this compound as a
vehicle for Isx-9 administration), as these authors did not
include a naı¨ve group in their study. This further highlights
the importance of including a naı¨ve group when studying
adult hippocampal neurogenesis. Since cyclodextrins are
poorly absorbed in the gut, administration of this vehicle
through the oral route may be an alternative to counteract
the putative toxicity of this compound (Stella and He,
2008). However, it is currently unknown whether a signif-
icant amount of Isx-9 can reach the brain when using HP-
b-CD as the vehicle if the drug is administered by oral
route. Thus, future studies are warranted to evaluate the
possibility of administering Isx-9 through the oral route
and/or using different vehicles (or lower doses of HP-b-
CD) to minimize the potential toxic effects associated with
this vehicle.
Nevertheless, regardless of the underlying cause of
the decreased cell proliferation and neuronal
commitment observed in the vehicle-treated group
(procedural stress and/or potential vehicle-induced
neurotoxicity), Isx-9 was able to completely prevent
these deficits, further corroborating the neurogenic
properties of this synthetic compound. Thus, the
development of synthetic molecules structurally and
functionally related to Isx-9 that possess a greater half-
life (thus reducing the need for frequent administration)
and better solubility than this compound may prove to
have therapeutic value for the treatment of conditions
associated with an increase in stress levels. Moreover,
based on the results reported here, we propose that a
naı¨ve group (not submitted to any manipulations) should
always be used in neurogenesis studies that involve the
repeated administration of compounds through i.p.
injections, as a control for the procedural stress and/or
vehicle-induced reduction in cell proliferation and
neuronal commitment.
To determine whether the reduction in hippocampal
cell proliferation and neuronal commitment induced by
procedural stress and/or vehicle-associated
neurotoxicity was accompanied by alterations in
circulating levels of CORT, we also determined the
levels of this stress-related hormone following the
14 days of vehicle or Isx-9 administration. CORT binds
to mineralocorticoid receptors (MR) and/or
glucocorticoid receptors (GR), which are ligand-driven
transcription factors that translocate to the nucleus
affecting gene transcription upon activation (Groeneweg
et al., 2012). Both these receptors are involved in the acti-
vation of several distinct signaling pathways, which may
Fig. 5. Effect of repeated administration of Isx-9 on serum corticos-
terone (CORT) levels. Circulating CORT levels were significantly
elevated in animals treated with Isx-9 for 14 days as compared to rats
treated with vehicle [(2-hydroxypropyl)-b-cyclodextrin]. Bars repre-
sent mean ± SEM. *
p < 0.05 in comparison with vehicle-treated
animals.
L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222 219](https://image.slidesharecdn.com/90d156e9-5ee3-4761-848d-fd7048fb8cb2-160809181804/85/ISX9-Karthik-Gopalakrishnan-8-320.jpg)
![stimulate (through MR activation) or suppress (through
GR activation) hippocampal cell proliferation (Anacker
et al., 2011). Since MR has ten-fold more affinity for
CORT, few GR binding sites are activated in baseline
conditions, but a significant change in its activation is
observed with an increase in CORT release (Datson
et al., 2008). Surprisingly however, despite the significant
reduction in the numbers of proliferating cells and imma-
ture neurons observed in vehicle-injected rats, we did
not find a significant alteration in CORT levels in these
animals. It may be possible that daily i.p. injections and
manipulations caused not only a reduction in hippocampal
neurogenesis (at the level of cell proliferation and neu-
ronal commitment) but also a dysregulation in the
hypothalamus-pituitary-adrenal (HPA) axis with a conse-
quent hypoactivation of the stress response. Such mech-
anism is commonly observed following exposure to
chronic stress (Miller et al., 2007; Mizoguchi et al.,
2008). On the other hand, we found a significant increase
in circulating CORT levels in animals that received Isx-9
for 14 days. This finding is in accordance with previous
studies that have also shown an increase in CORT with
well-known pro-neurogenic factors such as environmental
enrichment (Benaroya-Milshtein et al., 2004; Moncek
et al., 2004), voluntary physical exercise (Adlard and
Cotman, 2004), caloric restriction (Patel and Finch,
2002; Martin et al., 2006), and treatment with antidepres-
sants such as fluoxetine [as previously demonstrated by
us (Machado et al., 2012) and other groups (Duncan
et al., 1998; Weber et al., 2006)]. One possible explana-
tion for our findings is that similarly to these pro-
neurogenic factors (i.e., environmental enrichment, volun-
tary physical exercise, caloric restriction, and fluoxetine),
the repeated administration of Isx-9 may cause moderate
stress in the animals, leading to an adaptive response and
consequent activation of molecular mechanisms of neuro-
plasticity that outweigh the effects of increased CORT
levels on hippocampal progenitor cells. Additionally, it is
also known that Isx-9 exerts its effects on hippocampal
neurogenesis through Mef2 transcription factors
(Schneider et al., 2008; Petrik et al., 2012) and GR plays
a role in the regulation of the activity of these proteins
(Speksnijder et al., 2012). Therefore, further studies are
needed to verify if the increased CORT levels observed
in the present study may be a response to an enhanced
activation of the Mef2 signaling pathway.
CONCLUSION
Together, our results confirm that the pro-neurogenic
properties of Isx-9 previously reported in mice are also
observed in rats. In addition, in the present study we
also showed that this synthetic compound can up-
regulate cell proliferation and number of immature
neurons both in the dorsal and the ventral aspects of
the hippocampal DG, highlighting the potential beneficial
effects of Isx-9 on hippocampal structural plasticity.
Moreover, the increase in circulating CORT levels
observed at the end of the 14-day period of Isx-9
treatment did not impair the pro-neurogenic properties
of this synthetic compound.
While our findings suggest that the ability of Isx-9 to
reverse the reduction in cell proliferation and number of
immature neurons induced by procedural stress (i.e.,
repeated handling and i.p. injections) and/or vehicle
neurotoxicity results from its potent pro-neurogenic
properties, future studies are warranted to evaluate if
this synthetic compound can also reverse deficits in
adult hippocampal neurogenesis and induce functional
recovery (i.e., amelioration of hippocampal-dependent
behavioral deficits) in animal models of stress (i.e.,
restraint stress and chronic unpredictable stress) as well
as other models of mood disorders. Furthermore, the
development of other synthetic molecules structurally
and functionally related to Isx-9 that possess a greater
half-life (thus reducing the frequency of administration)
and better solubility than this compound may be of
clinical relevance as therapeutic strategies for
neurological conditions associated with impaired adult
hippocampal neurogenesis.
AUTHORS CONTRIBUTIONS AND DISCLOSURE
STATEMENT
L.B. performed most of the experiments and data analysis
and wrote the first draft of the manuscript. A.R.P. and J.G.
M. wrote the animal care protocols, provided assistance
with the experimental design, analysis and interpretation
of the data, as well as the drafting and revision of the
manuscript. N.F.O. and R.P.H. synthesized and purified
Isx-9 and drafted the document describing these
procedures. K.G. and S.K. participated in the
experimental protocols, particularly cell counting. A.L.R.,
J.W. and B.R.C. provided financial support and critical
analysis of the experimental design and the final results.
All authors have read and approved the final
manuscript. The authors declare no conflicts of interest.
Acknowledgments—L.B., J.G.M. and A.L.S.R. acknowledge
funding from the Science Without Borders funding program [Pro-
grama Cieˆncia Sem Fronteiras/ Conselho Nacional de Desen-
volvimento Cientı´fico e Tecnolo´gico (CNPq), Project
#403120/2012-8] of the Brazilian Federal Government. J.W.
gratefully acknowledges salary support from the Michael Smith
Foundation for Health Research (MSFHR) Career Scholar Pro-
gram and the Canada Research Chairs (CRC) Program, as well
as funding support from the Natural Sciences and Engineering
Research Council of Canada (NSERC). B.R.C. is supported by
grants from the Canadian Institutes of Health Research (CIHR),
NSERC, and the Canada Foundation for Innovation (CFI).
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ISX9 - Karthik Gopalakrishnan
- 1. ISX-9 CAN POTENTIATE CELL PROLIFERATION AND NEURONAL COMMITMENT IN THE RAT DENTATE GYRUS LUIS E. B. BETTIO, a,by ANNA R. PATTEN, ay JOANA GIL-MOHAPEL, a NATASHA F. O’ROURKE, c RONAN P. HANLEY, c SAMANTHA KENNEDY, a KARTHIK GOPALAKRISHNAN, a,d ANA LU´ CIA S. RODRIGUES, b JEREMY WULFF c AND BRIAN R. CHRISTIE a * a Division of Medical Sciences and UBC Island Medical Program, University of Victoria, Victoria, British Columbia, Canada b Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil c Department of Chemistry, University of Victoria, Victoria, British Columbia, Canada d Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada Abstract—Adult hippocampal neurogenesis can be modu- lated by various physiological and pathological conditions, including stress, affective disorders, and several neurologi- cal conditions. Given the proposed role of this form of struc- tural plasticity in the functioning of the hippocampus (namely learning and memory and affective behaviors), it is believed that alterations in hippocampal neurogenesis might underlie some of the behavioral deficits associated with these psychiatric and neurological conditions. Thus, the search for compounds that can reverse these deficits with minimal side effects has become a recognized priority. In the present study we tested the pro-neurogenic effects of isoxazole 9 (Isx-9), a small synthetic molecule that has been recently identified through the screening of chemical libraries in stem cell-based assays. We found that adminis- tration of Isx-9 for 14 days was able to potentiate cell prolif- eration and increase the number of immature neurons in the hippocampal DG of adult rats. In addition, Isx-9 treatment was able to completely reverse the marked reduction in these initial stages of the neurogenic process observed in vehicle-treated animals (which were submitted to repeated handling and exposure to daily intraperitoneal injections). Based on these results, we recommend that future neuroge- nesis studies that require repeated handling and manipula- tion of animals should include a naı¨ve (non-manipulated) control to determine the baseline levels of hippocampal cell proliferation and neuronal differentiation. Overall, these findings demonstrate that Isx-9 is a promising synthetic compound for the mitigation of stress-induced deficits in adult hippocampal neurogenesis. Future studies are thus warranted to evaluate the pro-neurogenic properties of Isx- 9 in animal models of affective and neurological disorders associated with impaired hippocampal structural plasticity. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: adult hippocampal neurogenesis, cell prolifera- tion, isoxazole 9 (Isx-9), immature neurons, stress. INTRODUCTION Neurogenesis in the adult brain results in the production of new neurons from a pool of progenitor cells in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). This form of structural plasticity can be modulated by a number of factors that include: exercise (van Praag et al., 1999a, 1999b); environmental enrichment (Kempermann et al., 1997); learning (Gould et al., 1999); and stress (Gould et al., 1998) and is thought to play a role in certain aspects of cognition, including hippocampal-dependent learning and memory (Gould, 1999; Kempermann, 2002) as well as affective (i.e., anxiety- and depressive-like) behaviors (Bannerman et al., 2004; Degroot and Treit, 2004; Engin and Treit, 2007). The hippocampus is one of the most malleable structures in the brain, and it can respond to external stimuli through structural and functional neuroplastic adaptations, a feature that can lead to beneficial or deleterious alterations in brain functioning (Sapolsky, 2003; McEwen and McEwen, 2008). Since this brain region has one of the highest concentrations of receptors for glucocorticoids, the hippocampus is particularly vul- nerable to the effects of stress, which in turn may have an inhibitory effect on hippocampal plasticity, namely adult neurogenesis (Kim and Diamond, 2002). Indeed, several studies have demonstrated that chronic exposure of rodents to corticosterone (CORT) inhibits hippocampal cell proliferation and differentiation (Wong and Herbert, 2006; Murray et al., 2008; Brummelte and Galea, 2010), an effect that seems to correlate with cognitive dysfunc- tion (Drapeau et al., 2003; Monje and Dietrich, 2012) and the pathogenesis of depressive disorders (Gregus et al., 2005; Zhao et al., 2008). Compounds that stimulate the generation of endogenous neural progenitors in the hippocampus may counteract the hippocampal neuronal http://dx.doi.org/10.1016/j.neuroscience.2016.06.042 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Address: Division of Medical Sciences, Island Medical Program, University of Victoria, Victoria, BC V8W 2Y2, Canada. Fax: +1-250-772-5505. E-mail address: brain64@uvic.ca (B. R. Christie). y These authors contributed equally to this work. Abbreviations: BrdU, 5-bromo-20 -deoxyuridine; CORT, corticosterone; DAB, 2,2-diaminobenzidine; DG, dentate gyrus; GR, glucocorticoid receptors; Isx-9, isoxazole 9; Mef2, myocyte enhancer factor-2; MR, mineralocorticoid receptors; NHS, normal horse serum; PFA, paraformaldehyde; SGZ, subgranular zone; TBS, Tris-buffered saline. Neuroscience 332 (2016) 212–222 212
- 2. loss and/or the development of cognitive deficits that are associated with certain neuropathological conditions. Thus, neurogenic drugs present potential therapeutic value for treatment of these disorders (Taupin, 2011). Screening analysis of chemical libraries in stem cell- based assays has identified several pro-neurogenic small molecules with therapeutic potential (Schneider et al., 2008; Pieper et al., 2010; Wurdak et al., 2010). Within this context, isoxazole 9 [Isx-9; N-cyclopropyl-5-(t hiophen-2-yl)isoxazole-3-carboxamide] was reported to influence stem-cell fate both in vitro and in vivo, namely by inducing a robust increase in neuronal differentiation. This effect seems to occur through the modulation of myo- cyte enhancer factor-2 (Mef2) (Schneider et al., 2008; Petrik et al., 2012), a family of transcription factors that plays a key role in the activation of genetic programs that control cell differentiation, proliferation, morphogenesis, survival and apoptosis (Potthoff and Olson, 2007). In a previous in vivo study, it was demonstrated that Isx-9 crosses the blood–brain barrier and is a safe pharmacological approach to increase neurogenesis in the SGZ of the hippocampal DG in adult mice (Petrik et al., 2012). However, despite being a promising syn- thetic neurogenic compound, our current knowledge on its in vivo properties is still limited. Thus, in the present study we confirmed and expanded the results initially reported by Petrik et al. (2012) and demonstrated the neurogenic effects of Isx-9 on cell proliferation and neu- ronal commitment in the adult rat hippocampal DG follow- ing repeated exposure to a commonly used laboratory procedure that is potentially associated with increased levels of stress (repeated intraperitoneal, i.p., injections). We found that Isx-9 was able to reverse the reduction in hippocampal cell proliferation and neuronal commitment found in vehicle-treated animals, further highlighting the neurogenic properties of this compound. Thus, the devel- opment of synthetic molecules structurally and function- ally related to Isx-9 that possess a greater half-life than this compound (thus reducing the frequency of adminis- tration) may prove to have therapeutic value for the treat- ment of conditions associated with an increase in stress levels. EXPERIMENTAL PROCEDURES Synthesis of Isx-9 Isx-9 was prepared according to the method of Schneider et al. with minor modifications (Fig. 1). All reactions were performed in oven- or flame-dried glassware, under a positive pressure of argon, unless otherwise indicated. Organic solutions were concentrated between 35 and 40 °C by rotary evaporation under vacuum. All reagents were used as received from commercial suppliers unless otherwise indicated. Commercial solvents were used as received with the following exceptions. Dichloromethane, tetrahydrofuran and toluene were dried by passage through a column of alumina in a commercial solvent purification system. Methanol was dried over activated 4 A˚ MS. 1 H chemical shifts are reported in parts per million (ppm, d scale) downfield from tetramethylsilane, and are referenced to residual protium in the NMR solvent (CDCl3: d 7.26; (CD3)2CO: d 2.05). Likewise, 13 C chemical shifts are referenced to the carbon resonances of the solvent (CDCl3: d 77.16; (CD3)2CO: d 29.85). Infrared spectra were collected using an FT-IR spectrometer. (Z)-Methyl 4-hydroxy-2-oxo-4-(thiophen-2-yl)but-3-en oate (1). To a stirred solution of 2-acetylthiophene (2.57 mL, 23.8 mmol) and dimethyloxylate (3.72 g, 31.5 mmol) in 125 mL dry toluene was added 30 mL of a solution of t-BuOK (1.0 M in THF, 30.0 mmol) dropwise at room temperature. Once addition was complete, the resultant slurry was stirred overnight at room temperature ($16 h). The reaction was quenched with 60 mL of 1 N HCl (aq) and the two phases were separated. The organic layer was washed with water and then brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was recrystallized from hexanes to afford compound 1 as a bright yellow powder in 86% yield. 1 H NMR (300 MHz, CDCl3): d 7.85 (dd, J = 3.8, 1.2 Hz, 1H), 7.72 (dd, J = 5.0, 1.2 Hz, 1H), 7.19 (dd, J = 5.0, 3.8 Hz, 1H), 6.93 (s, 1H), 3.94 (s, 3H); 13 C NMR (75 MHz, CDCl3) d 186.2 (C), 164.6 (C), 162.7 (C), 142.2 (C), 135.4 (CH), 132.8 (CH), 128.9 (CH), 99.8 (CH), 53.3 (CH3); IR (KBr, cmÀ1 ) 3114 (m), 3092 (m), 3076 (w), 2964 (m), 1751 (m), 1738 (s), 1611 (s), 1426 (s), 1250 (s), 1228 (s), 1121 (s), 1063 (m), 968 (m), 826 (m), 776 (s), 732 (s), 682 (m). Methyl 5-(thiophen-2-yl)isoxazole-3-carboxylate (2). N-Hydroxylamine hydrochloride (1.67 g, 24.0 mmol) was added to a stirring solution of ketone 1 (4.21 g, 19.8 mmol) in 100 mL of dry methanol and the reaction mixture was then heated to 60 °C for 12 h. The resultant solution was then concentrated under reduced pressure and the precipitated beige solid was resuspended in 100 mL of water. The resulting slurry was stirred at room temperature for 1 h before the product was collected by filtration through a sintered glass fritted funnel to yield compound 2 as a beige solid in quantitative yield. 1 H NMR (300 MHz, CDCl3): d 7.56 (dd, J = 3.8, 1.2 Hz, 1H), 7.50 (dd, J = 5.0, 1.2 Hz, 1H), 7.14 (dd, J = 5.1, 3.7 Hz, 1H), 6.78 (s, 1H), 3.99 (s, 3H); 13 C NMR (75 MHz, CDCl3) d 167.0 (C), 160.4 (C), 156.8 (C), 129.0 (CH), 128.4 (CH), 128.3 (C), 128.0 (CH), 99.7 (CH), 53.1 (CH3); IR (KBr, cmÀ1 ) 3134 (s), 3116 (m), 3083 (w), 2954 (m), 1731 (s), 1593 (s), 1476 (s), 1452 (s), 1275 (s), 1250 (s), 1138 (s), 1004 (s), 930 (s), 853 (s), 810 (s), 779 (s), 703 (s). 5-(Thiophen-2-yl)isoxazole-3-carboxylic acid (3). An aqueous solution of LiOH (1.0 M, 140 mL) was added to a stirred solution of ester 2 (16.9 g, 80.6 mmol) in 90 mL of THF and reacted at 50 °C for 3 h. The reaction mixture was cooled to room temperature and the aqueous layer was acidified to pH 1 (with 3 M HCl) and then extracted with ethyl acetate (2 Â 100 mL). The combined organic extracts were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to afford compound 3 as a yellow crystalline solid in 71% yield. 1 H NMR (300 MHz, (CD3)2CO): d 7.79 (dd, J = 5.1, 1.0 Hz, 1H), 7.76 (dd, J = 3.5, 1.2 Hz, 1H), 7.26 (dd, J = 5.1, 3.7 Hz, 1H), 7.06 (s, 1H); L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222 213
- 3. 13 C NMR (75 MHz, (CD3)2CO) d 167.6 (C), 160.9 (C), 158.3 (C), 130.4 (CH), 129.4 (CH), 129.1 (CH), 129.0 (C), 100.6 (CH); IR (KBr, cmÀ1 ) 3424 (br, w), 3132 (m), 3103 (m), 2882 (w), 1711 (s), 1686 (s), 1595 (s), 1443 (s), 1272 (s), 1249 (m), 1197 (m), 997 (m), 852 (m), 765 (m), 727 (s). N-Cyclopropyl-5-(thiophen-2-yl)isoxazole-3-carbox- amide (Isx-9) (Schneider et al., 2008). EDCÁHCl (5.39 g, 28.1 mmol) and HOBt (4.93 g, 32.2 mmol) were added sequentially to a stirred solution of acid 3 (4.49 g, 23.0 mmol) in dry dichloromethane (69 mL). The reaction mixture was stirred at room temperature for 15 min and cyclopropylamine (1.91 mL, 27.6 mmol) was then added dropwise to the reaction vessel. After 2 days, the reaction was concentrated under reduced pressure and the resi- due was resuspended between water and ethyl acetate (1:1). The two phases were separated and the aqueous layer was extracted with ethyl acetate four times. The combined organic extracts were washed with brine, con- centrated in vacuo, and purified by recrystallization (1:1 EtOH–water) to afford Isx-9 as a beige crystalline solid in 69% yield. 1 H NMR (300 MHz, CDCl3): d 7.53 (dd, J = 3.8, 1.2 Hz, 1H), 7.48 (dd, J = 5.0, 1.2 Hz, 1H), 7.14 (dd, J = 5.0, 3.5 Hz), 6.91 (br s, 1H), 6.82 (s, 1H), 2.94-2.85 (m, 1H), 0.92-0.84 (m, 2H), 0.71-0.64 (m, 2H); 13 C NMR (75 MHz, CDCl3) d 166.7 (C), 160.1 (C), 159.2 (C), 128.9 (CH), 128.6 (C), 128.3 (CH), 127.8 (CH), 98.9 (CH), 22.7 (CH), 6.8 (CH2); IR (KBr cmÀ1 ) 3368 (m), 3316 (s), 3154 (w), 3130 (m), 3102 (m), 3080 (m), 3012 (w), 1649 (s), 1594 (s), 1560 (s), 1453 (s), 1410 (s), 1272 (m), 1201 (m), 1179 (w), 1056 (m), 960 (m), 909 (m), 847 (s), 803 (s0, 718 (s), 706 (s), 566 (m). Animals Seventy-day-old male Sprague–Dawley rats (300 g; Charles River Laboratories, Montreal, Canada) were housed in pairs in clear polycarbonate cages (46 Â 24 Â 20 cm) with Carefresh contact bedding (Absorption Corp., Bellingham, WA, USA) for a 10-day- acclimation period following arrival to our animal care facility. Colony rooms were maintained at 21 °C, and on a 12-h light/dark cycle, with lights on at 7:00 AM. All rats were given ad libitum access to a regular chow diet (Lab Diets 5001; LabDiets, Richmond, IN, USA) and tap water. All protocols were performed in accordance with the Canadian Council for Animal Care and were approved by the Animal Care Committee of the University of Victoria. Following the acclimatization period, a total of 24 rats were assigned into three different experimental groups (n = 6–10 animals/group): naı¨ve, vehicle injection and Isx-9 injection. Animals were housed in isolation (i.e., one animal per cage). Drugs and treatment Isx-9 solution was prepared using (2-hydroxypropyl)-b-c yclodextrin (HP-b-CD; Sigma, St. Louis, MO, USA) as vehicle. The stock solution was prepared to a final concentration of 4 mg/ml Isx-9 and 30% (w/v) vehicle in sterile milliQ-purified H2O (Millipore Corp., Billerica, MA, USA). Vehicle or Isx-9 (20 mg/kg) were injected intraperitoneally (i.p.) once daily for 14 days (always at 9:00 AM). The dose was chosen based on a previous study (Petrik et al., 2012). 5-Bromo-20 -deoxyuridine (BrdU), a thymidine analog that is incorporated into the DNA of cells during S-phase of the cell cycle (Cooper-Kuhn and Kuhn, 2002), was used to label dividing cells. The BrdU solution (10 mg/ mL; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% sodium chloride (NaCl). On the 15th day (i.e., fol- lowing the 14-day period of Isx-9 treatment), vehicle- and Isx-9-treated animals received two doses (12 h apart; at 8:00 AM and 8:00 PM) of BrdU (150 mg/kg/injection; i. p.). Naı¨ve animals were not submitted to handling or i.p. injections during this period. On the 16th day, all animals were sacrificed by transcardial perfusion and their brains removed and processed for immunohistochemical analy- ses of hippocampal cell proliferation and number of imma- ture neuroblasts. Tissue processing Animals were deeply anesthetized with isoflurane (1–3% vapourizer; Abbott Laboratories, North Chicago, IL, USA) and transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde (PFA). The brains were removed and left in 4% PFA overnight at 4 °C and then transferred to 30% sucrose. Following saturation in sucrose, serial coronal sections were obtained on a Fig. 1. Synthesis of Isx-9. Isx-9 was prepared according to the method reported by Schneider et al. (2008). See text (Experimental procedures) for a detailed description of the synthesis protocol employed. 214 L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222
- 4. vibratome (Leica VT1000S, Nussloch, Germany) at 30- lm thickness. Sections were collected in a 1/6-section- sampling fraction and stored in a cryoprotectant solution [0.04 M Tris-buffered saline (TBS), 30% ethylene glycerol, 30% glycerol] at 4 °C. Immunohistochemistry One series of free-floating brain sections was processed for BrdU immunohistochemistry as previously described (Gil-Mohapel et al., 2011). Briefly, after thorough rinsing in 0.1 M TBS buffer (84 mM Tris–HCl, 16 mM Tris, 0.9% NaCl, pH = 7.4), the sections were incubated in 2 N HCl at 65 °C for 30 min to denature the DNA. The sec- tions were then pre-incubated for 1 h at room temperature (24 °C) in 5% normal horse serum (NHS) and 0.25% Tri- ton X-100 in 0.1 M TBS and then incubated for 48 h at 4 °C with a mouse monoclonal antibody against BrdU (1:60, M0744; Dako, Glostrup, Denmark) in TBS contain- ing 5% NHS. After incubation with a biotinylated horse anti-mouse IgG secondary antibody (1:200, BA-2001; Vector Laboratories, Burlingame, CA, USA) for 2 h at room temperature, the bound antibodies were visualized using an avidin-biotin-peroxidase complex system (Vec- tastain ABC Elite kit PK4000; Vector Laboratories) with 2,2-diaminobenzidine (DAB, DAB kit SK 4100; Vector Laboratories) as the chromogen. The sections were mounted onto 2% gelatin-coated microscope slides, dehydrated in a series of ethanol solutions of increasing concentrations followed by a 5-min incubation with a xylene substitute (CitriSolv; Fisher Scientific, Pittsburgh, PA, USA), and coverslipped with Permount mounting medium (Fisher Scientific). An adjacent series of brain sections was also processed for detection of the endogenous proliferative marker Ki-67, a nuclear protein that is expressed during all active phases of the cell cycle, but is absent from cells at rest (Scholzen and Gerdes, 2000), as previously described (Gil-Mohapel et al., 2011). Briefly, after thor- ough rinsing, the sections were incubated in 10 mM sodium citrate buffer (in 0.1 M TBS, pH = 6.0) at 95 °C for 5 min. This step was repeated twice to completely unmask the antigens. After quenching with 3% H2O2/10% methanol in 0.1 M TBS for 15 min and pre- incubating with 5% normal goat serum (NGS) for 1 h at room temperature, the sections were incubated for 48 h at 4 °C with a rabbit polyclonal primary antibody against Ki-67 (1:500, VP-K451; Vector Laboratories). After thor- ough rinsing, the sections were incubated for 2 h with the secondary antibody (biotin-conjugated goat anti- rabbit IgG, 1:200, BA-1000; Vector Laboratories) in 5% blocking solution at room temperature. The bound anti- bodies were detected using the avidin-biotin-peroxidase complex system (Vector Laboratories) with DAB (Vector Laboratories) as the chromogen. Brain sections were mounted onto microscope slides as described for BrdU immunolabelling (see details above). Finally, an additional series of brain sections was processed for NeuroD, a basic helix-loop-helix transcription factor involved in early neuronal maturation (Brunet and Ghysen, 1999; Miyata et al., 1999), as previ- ously described (Gil-Mohapel et al., 2011). Briefly, after quenching and pre-incubation with NHS at room temper- ature, the sections were incubated for 48 h at 4 °C with a goat anti-NeuroD primary antibody (1:200, SC-1084; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were then incubated for 2 h with the secondary antibody (biotin-conjugated horse anti-goat IgG, 1:200, BA-9500; Vector Laboratories) in 5% blocking solution at room temperature. The bound antibodies were detected using the avidin-biotin-peroxidase complex sys- tem (Vector Laboratories) with DAB (Vector Laboratories) as the chromogen. Brain sections were mounted onto microscope slides as described for BrdU immunolabelling (see details above). Morphological quantification by conventional microscopy All morphological analyses were performed on coded slides with the experimenter blinded to the identity of the brain sections (i.e., the animal), using an Olympus BX51 microscope equipped with 10Â, 40Â, and 100Â objectives (Olympus, Center Valley, PA, USA). Image Pro-Plus software (version 6.0 for WindowsTM, Media Cybernetic Inc., Silver Spring, MD, USA) and a Cool Snap HQ camera (Photometrics, Tucson, AZ, USA) were used for image capture. The total number of labeled cells present in all sections obtained from a single brain (i.e., animal) and containing the DG hippocampal sub-region were calculated, and that total number of cells were then used as the single data point from that respective animal. For each animal (i.e., brain), calculations were performed as follows: the total number of BrdU-, Ki-67- or NeuroD-immunopositive cells present in the SGZ of either the entire DG (from Bregma À2.30 to À6.04; approximately 20 coronal sections per brain), the dorsal DG (from Bregma À2.30 to À4.16; approximately 10 coronal sections per brain), or the ventral DG (Bregma À4.16 to À6.04; approximately 10 coronal sections per brain) sub- regions (Paxinos and Watson, 1986) were quantified by manually counting all DAB-positive cells present with 2– 3 cell diameters of the SGZ. Results were expressed as the total number of labeled cells in the DG hippocampal sub-region of each individual brain (i.e., animal) by multi- plying the average number of labeled cells/DG section by the total number of 30-lm-thick sections obtained from that respective animal and containing either the entire DG (125 slices), the dorsal DG (62 slices), or the ventral DG (63 slices). A single data point was entered for each individual animal and group averages were calculated as follows: P of total number of cells per brain (i.e., per animal)/total number of brains (i.e., animals). Images were processed with Adobe Photoshop 4.0 (Adobe Sys- tems, Mountain View, CA, USA). Only contrast enhance- ments and color level adjustments were made. CORT assay Blood tail samples were taken on the 14th day of treatment to assess CORT levels in the animals. Samples were stored at 4 °C overnight and then centrifuged for 30 min at 3000g to collect the serum L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222 215
- 5. (which was stored at À20 °C until processing). CORT levels were determined using an enzyme immunoassay kit (900-097, Assay Designs, Ann Habor, MI, USA) following the manufacturer’s instructions. Samples were run in duplicates. Briefly, the provided donkey anti- sheep IgG-coated 96-well plate was loaded with a CORT standard (in the range of 0–20,000 pg/ml) and the serum samples. An alkaline phosphatase conjugated to CORT and the polyclonal antibody against CORT was added to the wells and the plate was then gently shaken for 2 h at room temperature. Following three washes, wells were aspirated and p-nitrophenyl phosphate substrate solution was added and incubated for 1 h at room temperature to start the reaction of the alkaline phosphatase. The reaction was stopped by adding stop solution containing trisodium phosphate and CORT levels were determined at 405 nm with a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) and analyzed with the SoftMax Pro 5.2 software (Molecular Devices). CORT levels were calculated from the standard curve prepared for each plate and were expressed as ng/ml serum. Statistical analysis Statistical analyses were performed using the Statistica 7.1 analytical software (StatSoft Inc., Tulsa, OK, USA). Sample distributions presented equal variances and normal distributions (data not shown). Differences among experimental groups were compared using two- tailed unpaired Student’s t-tests or one-way analysis of variance (ANOVA) followed by Tukey’s multiple range post-hoc test when appropriate and correcting for multiple comparisons when necessary. A p value of less than 0.05 was considered to be statistically significant. RESULTS Effect of Isx-9 on hippocampal cell proliferation To evaluate the effect of Isx-9 on adult hippocampal cell proliferation, adult male Sprague–Dawley rats were treated with this compound (20 mg/kg/day; i.p.) for 14 days. Animals received two doses of the exogenous cell proliferation marker BrdU (150 mg/kg/i.p. injection; 12 h apart) on the 15th day and were sacrificed 12 h after the last injection. Their brains were then processed for immunohistochemistry against BrdU and endogenous markers of cell proliferation and neuronal commitment. Immunohistochemistry against BrdU revealed a significant increase in the number of BrdU+ cells in Isx-9-treated animals in comparison with vehicle- treated animals in the entire [t(12) = À4.81, p = 0.0007, Student’s t-test], dorsal [t(12) = À3.072 p = 0.011, Student’s t-test] and ventral [t(12) = À4.33 p = 0.001, Student’s t-test] hippocampal DG (Fig. 2). Since this exogenous cell proliferation marker is only incorporated into the DNA of dividing cells during the S- phase of the cell cycle, the exclusive use of BrdU may underestimate the number of cells undergoing cell division at any given time (and potentially fail to detect less robust differences in cell proliferation) (Eisch and Mandyam, 2007). Therefore, to confirm the results obtained with BrdU and further elucidate whether overall changes in cell proliferation (i.e., not just restricted to the number of cells in S-phase of the cell cycle) occurred with Isx-9 treatment, we also performed immunohisto- chemistry for the endogenous cell cycle protein Ki-67, which is expressed during all active phases of the cell cycle (Christie and Cameron, 2006). One-way ANOVA confirmed the proliferative effect of Isx-9 with a main effect of treatment observed in the entire hippocampal DG [F(2,21) = 8.87, p = 0.002]. Further post-hoc analysis revealed a reduction in cell proliferation in rats that received i.p. injections of vehicle during 14 days when compared with naı¨ve animals (which were not manipu- lated and did not receive any treatment during that time period) (p = 0.003, Tukey’s post-hoc test). Furthermore, this reduction in cell proliferation was prevented by Isx-9 treatment (p = 0.016, Tukey’s post-hoc test). Similar results were also found both in the dorsal and ventral aspects of the hippocampal DG [F(2,21) = 9.02, p = 0.002 and F(2,21) = 5.21, p = 0.001, respectively], where post-hoc analysis revealed that vehicle-injected rats presented a significant reduction in Ki-67+ cells both in the dorsal and ventral DG sub-regions (p = 0.004 and p = 0.016, respectively, Tukey’s post-hoc test). This effect was prevented by Isx-9 treatment in the dorsal aspect of the hippocampal DG (p = 0.008, Tukey’s post-hoc test) (Fig. 3). Fig. 2. Effect of repeated Isx-9 administration (20 mg/kg, i.p.) on DG cell proliferation as assessed by BrdU immunohistochemistry. A significant increase in the number of BrdU-positive cells was observed in rats treated for 14 days with Isx-9 in comparison with vehicle-treated animals in the entire, dorsal and ventral aspects of the hippocampal DG (A). Values are represented as mean ± SEM. Numbers of rats per experimental group are indicated in the respective bars. Representative photomicrographs of the effect of repeated treatment with vehicle or Isx-9 on the number of cells that incorporated the exogenous proliferation marker BrdU at 20Â (scale bar = 50 lm) and 40Â (scale bar = 10 lm) magnification (B). Examples of BrdU-positive cells are denoted by arrows. * p < 0.05 and ** p < 0.01 in comparison with vehicle-treated group (Student’s t- test). 216 L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222
- 6. Effect of Isx-9 on the number of immature neurons in the hippocampus The effect of Isx-9 on the number of immature neurons in the DG of the hippocampus was assessed by immunostaining slices with the immature neuronal marker NeuroD, as previously described by us (Boehme et al., 2011; Gil-Mohapel et al., 2011, 2013; Kannangara et al., 2014; Bettio et al., 2016). Statistical analysis demonstrated a significant main effect of treatment in the entire DG [F(2,20) = 8.12, p = 0.003]. Further post- hoc analysis revealed a reduction in the number of Neu- roD+ cells in the DG of rats treated with vehicle when compared with naı¨ve animals (which were not manipu- lated and did not receive any treatment during that time period) (p = 0.002, Tukey’s post-hoc test). This decrease in the number of immature neurons was prevented by Isx-9 treatment (p = 0.025, Tukey’s post-hoc test). Furthermore, evaluation of number of neuroblasts (i.e., expression of NeuroD) in the dorsal and ventral aspects of the hippocampal DG also revealed a significant main effect of treatment [F(2,20) = 5.36, p = 0.015 and F(2,20) = 8.81, p = 0.002, respectively]. Post-hoc analy- sis revealed that vehicle-treated rats showed a reduction in the number of immature neurons in both dorsal and ventral aspects of the hippocampal DG when compared with naı¨ve animals (p = 0.018 and p = 0.001, respec- tively, Tukey’s post-hoc test). Isx-9 treatment was able to prevent this reduction both in the dorsal (p = 0.046, Tukey’s post-hoc test) and ventral (p = 0.031, Tukey’s post-hoc test) aspects of the hippocampal DG (Fig. 4). Influence of Isx-9 on CORT levels To evaluate the levels of stress experienced by the animals as a consequence of the daily handling and injections procedure (Balcombe et al., 2004; Titterness and Christie, 2008), tail blood samples were obtained on day 14 (i.e., the last day of vehicle- or Isx-9 treatment) and circulating blood levels of CORT were measured. A one-way ANOVA revealed a significant main effect of Isx-9 treatment [F(2,21) = 4.13, p = 0.030]. Further post- hoc analysis revealed a significant increase in the circulat- ing levels of CORT in animals treated with Isx-9 when compared with vehicle-treated rats (p = 0.035, Tukey’s post-hoc test) (Fig. 5). DISCUSSION The present study confirmed the neurogenic properties of Isx-9 previously observed in mice (Petrik et al., 2012) and showed for the first time that repeated administration of this compound (for 14 days) is able to significantly increase stem cell proliferation and neuronal commitment in the DG of the hippocampus of adult rats and reverse the effects of repeated manipulation and injections on these initial stages of the neurogenic process. The hippocampus is a brain structure with a well- established role in the modulation of learning and memory (Whitlock et al., 2006; Bird and Burgess, 2008; Neves et al., 2008) as well as in mood regulation (Campbell and Macqueen, 2004; Ray et al., 2011). Since this structure is one of the few brain regions where new neurons are known to be born and to functionally integrate Fig. 3. Effect of repeated Isx-9 administration (20 mg/kg, i.p.) on DG cell proliferation as assessed by Ki-67 immunohistochemistry. Cell proliferation was significantly reduced in rats that received daily injections of vehicle [(2-hydroxypropyl)-b-cyclodextrin] for 14 days when compared to naı¨ve (i.e., non-manipulated) animals. This effect was reversed by Isx-9 treatment (20 mg/kg, i.p.) in the entire and dorsal aspect of the hippocampal DG (A). Values are represented as mean ± SEM. Animal numbers for each condition are indicated within the respective bar in the graphs. Representative photomicrographs of DG sections processed for Ki-67 immunohistochemistry from naı¨ve, vehicle- and Isx-9-treated rats at 20Â (scale bar = 50 lm) and 40Â (scale bar = 10 lm) magnification (B). Examples of Ki-67-positive cells are denoted by arrows. * p < 0.05 and ** p < 0.01 in comparison with naı¨ve rats, # p < 0.05 and ## p < 0.01 in comparison with vehicle-treated animals. L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222 217
- 7. into the pre-existing neuronal circuitry into adulthood, it is now believed that adult hippocampal neurogenesis plays a role in certain aspects of learning and memory as well as mood disorders (Giovanello et al., 2004; Dranovsky and Hen, 2006; Winocur et al., 2006; Becker and Wojtowicz, 2007; Gil-Mohapel et al., 2013). Furthermore, the hippocampus and its DG are not homogeneous struc- tures, having different patterns of gene expression and anatomical projections along their dorsal/ventral axis. The dorsal hippocampus (defined as 50% of hippocampal volume starting at the septal pole) communicates with brain regions associated with cognition, and is therefore primarily involved with processes of learning and memory. On the other hand, the ventral hippocampus (defined as 50% of the hippocampal volume starting at the temporal pole) is better situated to contribute to emotional responses, having an important role in motivational and affective behaviors (Bannerman et al., 2004; Fanselow and Dong, 2010). In the present study, we did not observe localized differences in the dorsal and ventral portions of the hippocampal DG with regard to the reduction in cell proliferation and number of neuroblasts induced by the repeated injection procedure and the reversion of this def- icit by Isx-9. This observation suggests that this com- pound has a broad neurogenic action, being able to potentiate both cell proliferation and neuronal commit- ment in both anatomical aspects of this structure. Although in our study we only used one marker of neuronal commitment (NeuroD, an helix-loop-helix transcription factor expressed in immature neurons or neuroblasts; Brunet and Ghysen, 1999; Miyata et al., 1999), the increase in cell proliferation induced by Isx-9 was mirrored by a similar increase in the number of NeuroD+ cells. Therefore, it is likely that the majority of proliferating cells observed in the hippocampal DG upon Isx-9 treatment are committed to the neuronal lineage. This conclusion is supported by the findings of Schneider et al. (2008), which indicate that Isx-9, through a mechanism involving N-methyl-D-aspartate (NMDA) receptor-induced Ca2+ signaling and Mef2 transcription, may indirectly activate NeuroD expression (Schneider et al., 2008). In addition, the study by Petrik et al. (2012) reinforces this conclusion by showing that Isx-9 leads to an increase in the proportion of BrdU/NeuN double-labeled cells but not BrdU/glial fibrillary acid pro- tein (GFAP) double-labeled cells (Petrik et al., 2012). Interestingly, Schneider et al. (2008) also demonstrated that Isx-9 not only induces robust neuronal differentiation, but also significantly blocks competing astrocytic differen- tiation in adult neural stem cells (Schneider et al., 2008). This effect was also observed in a recent study where this small synthetic molecule promoted the differentiation of neural stem cells but negatively affected oligodendrocyte precursor cells (OPCs) and endothelial progenitor cells (EPCs) (Koh et al., 2015). In our study, the Isx-9 solution was prepared using HP-b-CD, a vehicle that presents low toxicity and is commonly used for drug delivery to biological systems (Gould and Scott, 2005). Surprisingly, animals that received daily i.p. injections of vehicle during the 14-day treatment period demonstrated reduced levels of DG cell proliferation (as assessed with the cell proliferation mar- Fig. 4. Effect of repeated Isx-9 administration (20 mg/kg, i.p.) on DG neuronal differentiation as assessed by NeuroD immunohistochemistry. Animals injected with vehicle [(2-hydroxypropyl)-b-cyclodextrin] demonstrated a decrease in the number of NeuroD-positive neuroblasts in comparison with naı¨ve rats in the entire, dorsal and ventral aspects of the hippocampal DG. This impairment in neuronal differentiation was reversed by repeated Isx-9 administration in the entire DG (A). Values are represented as mean ± SEM. Numbers of animals per experimental group are indicated within the respective bars. Representative photomicrographs of hippocampal DG of naı¨ve, vehicle- and Isx-9-treated rats on the expression of the endogenous differentiation marker NeuroD at 20Â (scale bar = 50 lm) and 40Â (scale bar = 10 lm) magnification (B). Examples of NeuroD-positive cells are denoted by arrows. * p < 0.05 and ** p < 0.01 in comparison with naı¨ve rats, # p < 0.05 and ## p < 0.01 in comparison with vehicle-treated animals. 218 L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222
- 8. ker Ki-67; Fig. 2) and reduced number of neuroblasts (as assessed with the immature neuronal marker NeuroD; Fig. 3) when compared with animals that were not manip- ulated and did not receive any i.p. injections during the same period of time (i.e., naı¨ve rats). Given the low toxi- city of the vehicle used (Gould and Scott, 2005), we believe this decrease in hippocampal cell proliferation and in the number of immature neurons may be a conse- quence, at least in part, of the daily manipulations and injections these animals received (i.e., procedure- induced stress). In agreement, it is well-known that stress negatively impacts hippocampal cell proliferation and neuronal differentiation in mammals leading to a reduction in hippocampal volume (Joe¨ ls et al., 2007; Lee et al., 2009). The deleterious effects of stress on hippocampal structural plasticity (i.e., adult neurogenesis) have been repeatedly shown to occur in response to a large variety of stressors including subordination stress (Gould et al., 1997), resident-intruder stress (Gould et al., 1998), foot- shock (Malberg and Duman, 2003; Vollmayr et al., 2003), restraint stress (Pham et al., 2003; Bain et al., 2004), isolation (Dong et al., 2004), cold immobilization (Heine et al., 2004), cold swim (Lee et al., 2002; Heine et al., 2004), and predator odor (Tanapat et al., 1998; Falconer and Galea, 2003; Mirescu et al., 2004). Taking this into account, it is not surprising that in our study, 14 days of daily manipulations and i.p. injections (which, besides being a mildly painful procedure also involves brief periods of restraint) might have been stressful enough to cause a significant reduction in adult hip- pocampal cell proliferation and neuronal survival and/or differentiation. On the other hand, despite the low toxicity of the vehicle used to prepare the Isx-9 solution (HP-b-CD) (Gould and Scott, 2005), some studies have raised con- cerns regarding its safety since there is evidence for hematological and histopathological alterations in rats that received HP-b-CD through the intravenous route (Gould and Scott, 2005; Kantner and Erben, 2012). Regarding its effects on the central nervous system, var- ious studies have shown that chronic administration of HP-b-CD was not associated with neurotoxic effects when administered either systemically (Yao et al., 2012; Matsuo et al., 2013; Walenbergh et al., 2015) or through the intracerebroventricular route (Aqul et al., 2011; Matsuo et al., 2014). However, since the HP-b-CD-induced histopathological changes observed in previous studies (Gould and Scott, 2005; Kantner and Erben, 2012) were found with HP-b-CD doses lower than those used in the present study, we cannot rule out the possibility that vehicle-induced neurotoxicity might underlie, at least in part, the impairment on cell proliferation and/or neuronal commitment observed in the present study in the vehicle-treated group. In addition, the potential neurotox- icity of HP-b-CD might have been overlooked in the study by Petrik et al. (2012) (who also used this compound as a vehicle for Isx-9 administration), as these authors did not include a naı¨ve group in their study. This further highlights the importance of including a naı¨ve group when studying adult hippocampal neurogenesis. Since cyclodextrins are poorly absorbed in the gut, administration of this vehicle through the oral route may be an alternative to counteract the putative toxicity of this compound (Stella and He, 2008). However, it is currently unknown whether a signif- icant amount of Isx-9 can reach the brain when using HP- b-CD as the vehicle if the drug is administered by oral route. Thus, future studies are warranted to evaluate the possibility of administering Isx-9 through the oral route and/or using different vehicles (or lower doses of HP-b- CD) to minimize the potential toxic effects associated with this vehicle. Nevertheless, regardless of the underlying cause of the decreased cell proliferation and neuronal commitment observed in the vehicle-treated group (procedural stress and/or potential vehicle-induced neurotoxicity), Isx-9 was able to completely prevent these deficits, further corroborating the neurogenic properties of this synthetic compound. Thus, the development of synthetic molecules structurally and functionally related to Isx-9 that possess a greater half- life (thus reducing the need for frequent administration) and better solubility than this compound may prove to have therapeutic value for the treatment of conditions associated with an increase in stress levels. Moreover, based on the results reported here, we propose that a naı¨ve group (not submitted to any manipulations) should always be used in neurogenesis studies that involve the repeated administration of compounds through i.p. injections, as a control for the procedural stress and/or vehicle-induced reduction in cell proliferation and neuronal commitment. To determine whether the reduction in hippocampal cell proliferation and neuronal commitment induced by procedural stress and/or vehicle-associated neurotoxicity was accompanied by alterations in circulating levels of CORT, we also determined the levels of this stress-related hormone following the 14 days of vehicle or Isx-9 administration. CORT binds to mineralocorticoid receptors (MR) and/or glucocorticoid receptors (GR), which are ligand-driven transcription factors that translocate to the nucleus affecting gene transcription upon activation (Groeneweg et al., 2012). Both these receptors are involved in the acti- vation of several distinct signaling pathways, which may Fig. 5. Effect of repeated administration of Isx-9 on serum corticos- terone (CORT) levels. Circulating CORT levels were significantly elevated in animals treated with Isx-9 for 14 days as compared to rats treated with vehicle [(2-hydroxypropyl)-b-cyclodextrin]. Bars repre- sent mean ± SEM. * p < 0.05 in comparison with vehicle-treated animals. L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222 219
- 9. stimulate (through MR activation) or suppress (through GR activation) hippocampal cell proliferation (Anacker et al., 2011). Since MR has ten-fold more affinity for CORT, few GR binding sites are activated in baseline conditions, but a significant change in its activation is observed with an increase in CORT release (Datson et al., 2008). Surprisingly however, despite the significant reduction in the numbers of proliferating cells and imma- ture neurons observed in vehicle-injected rats, we did not find a significant alteration in CORT levels in these animals. It may be possible that daily i.p. injections and manipulations caused not only a reduction in hippocampal neurogenesis (at the level of cell proliferation and neu- ronal commitment) but also a dysregulation in the hypothalamus-pituitary-adrenal (HPA) axis with a conse- quent hypoactivation of the stress response. Such mech- anism is commonly observed following exposure to chronic stress (Miller et al., 2007; Mizoguchi et al., 2008). On the other hand, we found a significant increase in circulating CORT levels in animals that received Isx-9 for 14 days. This finding is in accordance with previous studies that have also shown an increase in CORT with well-known pro-neurogenic factors such as environmental enrichment (Benaroya-Milshtein et al., 2004; Moncek et al., 2004), voluntary physical exercise (Adlard and Cotman, 2004), caloric restriction (Patel and Finch, 2002; Martin et al., 2006), and treatment with antidepres- sants such as fluoxetine [as previously demonstrated by us (Machado et al., 2012) and other groups (Duncan et al., 1998; Weber et al., 2006)]. One possible explana- tion for our findings is that similarly to these pro- neurogenic factors (i.e., environmental enrichment, volun- tary physical exercise, caloric restriction, and fluoxetine), the repeated administration of Isx-9 may cause moderate stress in the animals, leading to an adaptive response and consequent activation of molecular mechanisms of neuro- plasticity that outweigh the effects of increased CORT levels on hippocampal progenitor cells. Additionally, it is also known that Isx-9 exerts its effects on hippocampal neurogenesis through Mef2 transcription factors (Schneider et al., 2008; Petrik et al., 2012) and GR plays a role in the regulation of the activity of these proteins (Speksnijder et al., 2012). Therefore, further studies are needed to verify if the increased CORT levels observed in the present study may be a response to an enhanced activation of the Mef2 signaling pathway. CONCLUSION Together, our results confirm that the pro-neurogenic properties of Isx-9 previously reported in mice are also observed in rats. In addition, in the present study we also showed that this synthetic compound can up- regulate cell proliferation and number of immature neurons both in the dorsal and the ventral aspects of the hippocampal DG, highlighting the potential beneficial effects of Isx-9 on hippocampal structural plasticity. Moreover, the increase in circulating CORT levels observed at the end of the 14-day period of Isx-9 treatment did not impair the pro-neurogenic properties of this synthetic compound. While our findings suggest that the ability of Isx-9 to reverse the reduction in cell proliferation and number of immature neurons induced by procedural stress (i.e., repeated handling and i.p. injections) and/or vehicle neurotoxicity results from its potent pro-neurogenic properties, future studies are warranted to evaluate if this synthetic compound can also reverse deficits in adult hippocampal neurogenesis and induce functional recovery (i.e., amelioration of hippocampal-dependent behavioral deficits) in animal models of stress (i.e., restraint stress and chronic unpredictable stress) as well as other models of mood disorders. Furthermore, the development of other synthetic molecules structurally and functionally related to Isx-9 that possess a greater half-life (thus reducing the frequency of administration) and better solubility than this compound may be of clinical relevance as therapeutic strategies for neurological conditions associated with impaired adult hippocampal neurogenesis. AUTHORS CONTRIBUTIONS AND DISCLOSURE STATEMENT L.B. performed most of the experiments and data analysis and wrote the first draft of the manuscript. A.R.P. and J.G. M. wrote the animal care protocols, provided assistance with the experimental design, analysis and interpretation of the data, as well as the drafting and revision of the manuscript. N.F.O. and R.P.H. synthesized and purified Isx-9 and drafted the document describing these procedures. K.G. and S.K. participated in the experimental protocols, particularly cell counting. A.L.R., J.W. and B.R.C. provided financial support and critical analysis of the experimental design and the final results. All authors have read and approved the final manuscript. The authors declare no conflicts of interest. Acknowledgments—L.B., J.G.M. and A.L.S.R. acknowledge funding from the Science Without Borders funding program [Pro- grama Cieˆncia Sem Fronteiras/ Conselho Nacional de Desen- volvimento Cientı´fico e Tecnolo´gico (CNPq), Project #403120/2012-8] of the Brazilian Federal Government. J.W. gratefully acknowledges salary support from the Michael Smith Foundation for Health Research (MSFHR) Career Scholar Pro- gram and the Canada Research Chairs (CRC) Program, as well as funding support from the Natural Sciences and Engineering Research Council of Canada (NSERC). B.R.C. is supported by grants from the Canadian Institutes of Health Research (CIHR), NSERC, and the Canada Foundation for Innovation (CFI). 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