Isoxazole 9 | Gw4869

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

*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: [email protected] (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.

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.
© 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

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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. 1H 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, 13C 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. 1H 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); 13C 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, cm—1) 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. 1H 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); 13C 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, cm—1) 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 (2 100 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. 1H 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);

214

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.

13C 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, cm—1) 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). EDC HCl (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. 1H 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); 13C 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 cm—1) 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 24 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

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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 10 , 40 , and 100 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 2.30 to 6.04; approximately 20 coronal sections per brain), the dorsal DG (from Bregma 2.30 to 4.16; approximately 10 coronal sections per brain), or the ventral DG (Bregma 4.16 to 6.04; 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: 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 20 °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) = 4.81, p = 0.0007, Student’s t-test], dorsal [t(12) = 3.072 p = 0.011, Student’s t-test] and ventral [t(12) = 4.33 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

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 20 (scale bar = 50 lm) and 40 (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).

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).

L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222 217

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 20 (scale bar = 50 lm) and 40 (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.

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

218 L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222

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 20 (scale bar = 50 lm) and 40 (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.

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-

L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222 219

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.

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

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

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

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

220 L. E. B. Bettio et al. / Neuroscience 332 (2016) 212–222

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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(Accepted 23 June 2016)
(Available online 29 June 2016)