Background: Previous studies have shown that a low dose of scopolamine produces rapid-acting antidepressant-like actions in rodents. Understanding the mechanisms underlying this effect and the dose-dependent variations of drug responses remains an important task. L-type voltage-dependent calcium channels were found to mediate rapid-acting antidepressant effects of certain medications (e.g., ketamine). Therefore, it is of great interest to determine the involvement of L-type voltage- dependent calcium channels in the action of scopolamine. Methods: Herein, we investigated the mechanisms underlying behavioral responses to various doses of scopolamine in mice to clarify the involvement of L-type voltage-dependent calcium channels in its modes of action. Open field test, novel object recognition test, and forced swimming test were performed on mice administered varied doses of scopolamine (0.025, 0.05, 0.1, 1, and 3 mg/kg, i.p.) alone or combined with L-type voltage-dependent calcium channel blocker verapamil (5 mg/kg, i.p.). Then, the changes in brain-derived neurotrophic factor and neuropeptide VGF (nonacronymic) levels in the hippocampus and prefrontal cortex of these mice were analyzed. Results: Low doses of scopolamine (0.025 and 0.05 mg/kg) produced significant antidepressant-like effects in the forced swimming test, while higher doses (1 and 3 mg/kg) resulted in significant memory deficits and depressive-like behaviors. Moreover, the behavioral changes in responses to various doses may be related to the upregulation (0.025 and 0.05 mg/kg) and downregulation (1 and 3 mg/kg) of brain-derived neurotrophic factor and VGF in the hippocampus and prefrontal cortex in mice. We further found that the rapid-acting antidepressant-like effects and the upregulation on brain-derived neurotrophic factor and VGF produced by a low dose of scopolamine (0.025 mg/kg) were completely blocked by verapamil. Conclusions: These results indicate that L-type voltage-dependent calcium channels are likely involved in the behavioral changes in response to various doses of scopolamine through the regulation of brain-derived neurotrophic factor and VGF levels. Keywords: L-type voltage-dependent calcium channel, scopolamine, brain-derived neurotrophic factor, neuropeptide VGF, cognitive deficits and depression-like behaviors Received: May 23, 2017; Revised: August 18, 2017; Accepted: August 23, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 175 provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/175/4093880 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 176 | International Journal of Neuropsychopharmacology, 2018 Significance Statement Previous studies demonstrate that a low dose of nonselective muscarinic receptor antagonist scopolamine produces rapid anti - depressant actions relative to conventional antidepressants, while high doses of this drug result in potential memory impair - ment. Herein, we demonstrate that low and high doses of scopolamine produce different effects on depression-like and cognitive behaviors along with opposite-directional changes in BDNF and VGF levels in the hippocampus and prefrontal cortex. We further reveal that L-VDCC blocker verapamil significantly blocked the effects of low-dose scopolamine, suggesting that L-VDCC may be involved in the changes of behavior as well as BDNF and VGF levels produced by scopolamine treatment. Introduction Conventional antidepressants lack efficacy in many patients Materials and Methods (treatment-resistant depression) and generally take weeks to produce a full therapeutic response in others. This is particu - Animals larly concerning given the high suicide risk in major depressive Adult male C57BL/6J mice (10–12 weeks old upon arrival) were disorder (MDD) (Trivedi et al., 2006 ). Previous studies have dem- born and reared in the animal facility of Ningbo University onstrated that specific drugs such as ketamine are rapidly acting Medical School, China. All animals were maintained at 22°C antidepressants for resistant MDD patients ( Bartoli et al., 2017 ; ± 3°C and 60% ± 5% relative humidity under a 12-hour-light/-dark Duncan et al., 2017). Recently, clinical ( Janowsky et al., 1972 ; cycle (lights on at 7:00 am) with ad libitum access to food and Furey et al., 2015 ) and preclinical ( Podkowa et al., 2016 ; Martin water. All procedures involving animals were conducted follo - w et al., 2017 ) studies suggest that scopolamine was also reported ing the National Institute of Health Guidelines for the Care and to produce rapid and long-lasting antidepressant effects and Use of Laboratory Animals (NIH Publications No. 80-23, revised has shown similarities in the underlying mechanisms with ket - 1996) as well as the European Community Council Directive amine. However, scopolamine antagonizes muscarinic receptors for the Care and Use of Laboratory Animals of 22 September and may cause cognitive deficits at high doses ( Klinkenberg and 2010 (2010/63/EU). All the experiments were approved by the Blokland, 2010 ). The current study therefore aims to reveal the Institutional Animal Care and Use Committee of the Medical mechanisms underlying the varied effects of scopolamine at School of Ningbo University. different doses on cognitive changes and depressive-like beha- v iors in mice. Previous neuropharmacology studies indicate that brain Drugs derived neurotrophic factor (BDNF) and neuropeptide VGF (non - Scopolamine hydrochloride (Sigma-Aldrich) and verapamil acronymic) related signaling pathways play important roles in hydrochloride were dissolved in 0.9% saline and injected i.p. the action of multiple antidepressants. For example, murine with an injection volume of 10 mL/kg body weight at doses of studies have demonstrated the putative roles of BDNF and neu - 0.025, 0.05, 0.1, 1, and 3 mg/kg. Control groups received i.p. saline ropeptide VGF (nonacronymic) in the prefrontal cortex and/or injections with matched volumes. The experimental regimen is hippocampus in the rapid-acting antidepressant-like actions of shown in Figure 1. To explore the behavioral changes induced ketamine (Li et al., 2010 ; Duman and Aghajanian, 2012; Voleti by scopolamine, mice were grouped and received a single injec - et al., 2013 ; Pazini et al., 2016 ; Girgenti et al., 2017 ) and GLYX-13 tion of the drug at various doses (0.025, 0.05, 0.1, 1, and 3 mg/kg, (Lu et al., 2014). Previous mechanistic studies have shown that i.p.). After 24 hours, behavioral changes were examined using the rapid onset of antidepressant-like effect at lower doses of the open field test (OFT), novel object recognition test (NORT), scopolamine shares the same signaling cascades with ketamine and forced swimming test (FST) (Figure 1A). To test the involv- e by activating glutamate cycling activation and the mammalian ment of L-VDCC in the rapid-acting antidepressant-like effects target of rapamycin complex 1 ( Chowdhury et al., 2017 ; Wohleb of scopolamine, L-VDCC blocker verapamil was administered 60 et al., 2017 ; Voleti et al., 2013 ). Therefore, it was hypothesized minutes before treatment of scopolamine (0.025, 1, and 3 mg/ that ketamine-associated BDNF and/or VGF pathways might kg, i.p.) or saline (i.p.) on day 1, and then the OFT, NORT, and also mediate the action of scopolamine. Indeed, our recent FST were conducted from day 2 to day 5 successively ( Figure 1B). study suggested that BDNF/tropomyosin-related kinase receptor B (TrkB) signaling likely mediated antidepressant-like activities of muscarinic acetylcholine receptor antagonists ( Zhou et al., Behavioral Tests 2017), supporting the above hypothesis. Notably, the stimulation of L-type voltage-dependent calcium channel (L-VDCC) plays a OFT and NORT critical role in the release of BDNF ( Jourdi et al., 2009 ) and the OFT was performed to estimate the possible effects of drug rapid-acting antidepressant effects of ketamine ( Li et al., 2010 ; treatment on locomotor activity in mice. An apparatus made Duman and Aghajanian, 2012; Voleti et al., 2013 ; Pazini et al., of a white Plexiglas box (50 × 50 × 39 cm) with the floor divided 2016; Girgenti et al., 2017 ). However, whether L-VDCC is involved into 4 identical squares in a dim room was used for the test, in scopolamine’s regulation of BDNF and neuropeptide VGF and mice were habituated to an empty apparatus for 5 minutes (nonacronymic) remains unknown. Herein, we sought to investi - to test locomotor activity. Specifically, each mouse was placed gate whether pretreatment with verapamil, a phenylakylamine individually into the center of the arena and free exploration calcium channel antagonist, can modulate behavioral changes was recorded. The number of rearings (forepaws elevated from induced by various doses of scopolamine and neurotrophins the floor) and line crossings (with all 4 paws placed into a new (e.g., BDNF and VGF) in the hippocampus and prefrontal cortex square) were considered to be an index of exploratory behaviors. of mice. After each trial, the instrument was cleaned with 1% ethanol. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/175/4093880 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Yu et al. | 177 Figure 1. Schematic timeline of drug administration and behavioral testing. (A) Experimental procedure for the various scopolamine dose groups and cognitive and depression-like behaviors in the mice. (B) Experimental procedure for the assessment of the effects of L-type voltage-dependent calcium channel (L-VDCC) blocker verapamil in the regulation of behavioral changes and brain derived neurotrophic factor (BDNF) and VGF levels produced by various doses of scopolamine. Twenty-four hours after the habituation and OFT, the NORT conducted in a sound-attenuated room eliminated by white was conducted to quantify intact recognition memory of the light (40 luminous flux). Briefly, mice were placed individu - mice. This test is based on the tendency of mice to spend more ally in a clear plastic cylinder (height: 25 cm; diameter: 10 cm) time exploring a novel object than a familiar one. First, an containing 10 cm of fresh water at 23°C ± 2°C for 6 minutes, acquisition trial was conducted by leaving the animals in the and the duration of immobility was scored during the last 4 apparatus containing 2 identical objects (A and A’). Twenty-four minutes. Immobility was recognized when a mouse stopped hours later, recognition memory was evaluated, and a differ - struggling and continued with only slight movements to keep ent pair of dissimilar objects (a familiar and a novel one; A and its head above water. B, respectively) was presented. Each mouse was placed in the apparatus facing the wall and allowed to explore the objects Western-Blot Analysis for 5 minutes, after which the mouse was returned to its home Total proteins of the hippocampal and prefrontal cortex -tis cage. Their behavior was recorded by a video camera mounted sues of each mice (n = 3/group) were extracted using ice-cold vertically above the test arena and analyzed using appropri - radio immunoprecipitation assay lysis buffer (Pierce) containing ated video-tracking software (Duoyi). Object exploration was 50 mM Tris-HCl (pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium defined as when the mouse touched or sniffed the object from deoxycholate, 0.1% SDS; Upstate). The lysates were then cen - no more than 2 cm away, and the duration spent exploring each trifuged (15 000 × g, 30 minutes, 4°C), and the protein concen - object was scored by an observer blinded to the treatment group. tration of each sample lysate was determined using the BCA Exploration duration recorded was converted to a recognition kit (Thermo Scientific). The sample lysates (20 μg total pr-o index (RI) defined as RI = Tn/(Tn + Tf), where Tn is time spent tein) were separated on 10% SDS-polyacrylamide gel electr - o exploring the novel object, and Tf is the time spent exploring the phoresis gels and transferred to PVDF membranes (0.22 μm; familiar object. Between trials, the apparatus was cleaned with Millipore). Membranes were then incubated with anti-BDNF 1% ethanol solution to hide animal clues. The light and sound (1:800; Millipore), anti-VGF (1:1000; Cell Signaling), and rabbit inside the apparatus were maintained at a minimum level to anti-β-actin (1:2000; Millipore) at 4°C overnight. The membranes reduce anxiety behavior induced by environmental factors. were then incubated with Alexa Fluor 700 (for VGF) or 800 (for BDNF and β-actin)-conjugated antibody (1:10 000; Invitrogen) for FST 60 minutes. Target bands were detected and quantified using a The FST is one of the most commonly used assays for the fluorescence scanner (Odyssey Infrared Imaging System, LI-COR study of depressive-like behavior in rodents. The FST was Biotechnology). Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/175/4093880 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 178 | International Journal of Neuropsychopharmacology, 2018 Figure 2. Various doses of scopolamine-induced behavioral changes in mice. To measure general locomotor activity, we used the open field test (OFT). Animals were placed in a novel environment during a 5-minute session. (A) Line crossings and (B) rearing measurement of total distance traveled in time segments of 5 minutes. (C) Novel object recognition test (NORT). 24 hours after acquisition trial, the recognition index was measured while the animals were allowed to explore the familiar and novel objects for 5 minutes. (D) The increase in time spent immobile during the forced swimming test (FST) was also defined as depression-like behavior. Data are expressed as the mean ± SEM (n = 9/group); comparisons were made using the 1-way ANOVA followed by Newman-Keuls posthoc test. Significantly different from vehicle-treated group (saline plus saline). P ** < .01 for the drug-treatment group vs the control group. Immunohistochemistry slides, and coverslipped with ProLong (Invitrogen). The images were captured using the Olympus system and analyzed with Immunohistochemistry was performed to quantify the densi- ImageJ software. ties of VGF and BDNF (the cells showing positive staining). At killing time point, animals (n = 5/group) under pentobarbital Statistical Analysis anesthesia were transcardially perfused with 250 mL cold saline solution followed by 250 mL of 4% paraformaldehyde in 0.1 M All measurements were performed by an independent investiga - phosphate-buffered saline (PBS, pH 7.4). Brains were removed tor blinded to the experimental conditions. Data are presented and postfixed in the same fixative overnight and stored in 10%, as the mean ± SEM. One-way ANOVA followed by Newman- 20%, and 30% sucrose solution at 4 C for dehydration. The brains Keuls posthoc test using GraphPad Prism software (Version 6.0, were then quenched in cold N-hexane at −60C for 20 seconds Prism software for PC) was performed to analyze the data, and and stored at −80 C until required. Serial coronal sections of the P < .05 was considered significant. prefrontal cortex and hippocampus (30 μm thick; Paxinos and Watson, 2005) were collected on a cryostat (Leica). Free-floating sections were permeabilized with 0.2% TritonX-100 in PBS for Results 15 minutes, blocked in PBS containing 5% donkey serum for 1 hour at room temperature, and then incubated with anti-VGF Effects of Different Doses of Scopolamine on (1:400, Abcam) and anti-BDNF (1:800, Abcam) antibodies over - Locomotor Activity, Recognition, and Depression- night at 4C. The next day, all slices were rinsed and washed in Like Behaviors in Mice the PBS and then incubated with fluorescent secondary antibod - ies, donkey anti rabbit conjugated with Alexa Fluor 488 (1:1000; The OFT was conducted first to explore whether various doses of scopolamine (0.025, 0.05, 0.1, 1, and 3 mg/kg, i.p.) pro- Invitrogen), and donkey anti goat conjugated with Alexa Fluor 594 (1:1000; Invitrogen) for 1 hour at room temperature. DNA duced alterations of locomotor activity in mice. We found that scopolamine treatments had no effect on line crossings (nuclei) was stained with DAPI for 15 minutes, mounted onto Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/175/4093880 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Yu et al. | 179 [F (5, 48) = 0.08283, P = .9946; Figure 2A] or rearings [F (5, 48) = 0.5072, Effects of Different Doses of Scopolamine on the P = .7694; Figure 2B] compared with the saline-treated group, Levels of BDNF and VGF in the Murine Hippocampus indicating that behavioral changes observed in the subsequent and Prefrontal Cortex NORT and FST were not attributed to alterations in locomotor As shown in Figure 3, high doses of scopolamine significantly activity. Following the OFT, NORT was conducted to examine decreased the expression of BDNF in the hippocampus [F (5, the novel object recognition. We saw significant reduction of the 24) = 34.43, 0.1 mg/kg, P < .01, 1 mg/kg, P < .01 and 3 mg/kg, P < .01; object recognition index in the retention session in mice treated Figure 3B] and prefrontal cortex [F (5, 24) = 19.50, 1 mg/kg, P < .05 with 1 and 3 mg/kg scopolamine compared with the saline- and 3 mg/kg, P < .05; Figure 3E] compared with the saline treat - treated group [F (5, 48) = 8.613, P < .01; Figure 2C], suggesting that ment. However, the levels of BDNF in the hippocampus [F (5, scopolamine at these 2 doses induced cognitive impairment. 24) = 34.43, 0.025 mg/kg, P < .01, 0.05 mg/kg, P < .01; Figure 3B] However, no significant alterations in cognition were observed and prefrontal cortex [F (5, 24) = 19.50, 0.025 mg/kg, P < .05 and in these 2 groups during the training session (data not shown). 0.05 mg/kg, P < .05; Figure 3E] were significantly increased by In the FST for examination of depression-like behaviors, mice low doses of scopolamine treatment compared with the saline treated with low doses of scopolamine (0.025 and 0.05 mg/kg, i.p .) treatment. demonstrated significantly shorter periods of immobility than Consistent with the changes in BDNF, the levels of VGF were those treated with saline [F (5, 48) = 28.69, P < .01 for both doses] also significantly decreased by high doses of scopolamine in (Figure 2D), indicating antidepressant-like effects of scopolam - the hippocampus [F (5, 24) = 25.05, 1 mg/kg, P < .01 and 3 mg/kg, ine at these 2 doses in mice. However, high doses scopolamine (1 P < .01; Figure 3C] and prefrontal cortex [F (5, 24) = 24.06, 1 mg/kg, and 3 mg/kg, i.p.)-treated mice exhibited significantly increased P < .01 and 3 mg/kg, P < .05; Figure 3F] compared with the saline- immobility time compared with the saline-treated mice, thereby treated mice. In contrast, low doses of scopolamine resulted in showing depression-like behaviors [F (5, 48) = 28.69, P < .01 for a significant increase in VGF expression in the hippocampus both doses] (Figure 2D). Figure 3. Effects of various doses of scopolamine on the brain derived neurotrophic factor (BDNF) and VGF levels in brain regions. (A) and (D) representative imm- uno blots of BDNF and VGF detected by western blotting with tissues from the hippocampus (A) and prefrontal cortex (D); the panels (B,C,E,F) are the quantification of the immunoblotting bands of BDNF (B,E) and VGF (C,F) in the hippocampus or prefrontal cortex of the mice. The data are expressed as the mean ± SEM (n = 5/group). * P < .05, **P < .01, compared with control (saline plus saline) group. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/175/4093880 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 180 | International Journal of Neuropsychopharmacology, 2018 [F (5, 24) = 25.05, 0.025 mg/kg, P < .05, 0.05 mg/kg, P < .05; Figure 3B] Inhibition of L-VDCC Blocked the Rapid Onset of and prefrontal cortex [F (5, 24) = 24.06, 0.025 mg/kg, P < .05, Antidepressant-Like Effects by Scopolamine at Low 0.05 mg/kg, P < .05; Figure 3F] compared with the saline group. Doses in Mice The expression levels of VGF and BDNF in the prefrontal cortex To determine the involvement of L-VDCC in behavioral changes (Figure 4) and hippocampus ( Figure 5) of mice were visualized induced by various doses of scopolamine, mice were divided using immunofluorescence. Co-staining of VGF with BDNF in into several groups and treated as follows: (1) mice treated with prefrontal cortex and hippocampus revealed overlapped colo - saline (vehicle for verapamil, i.p.) plus saline (vehicle for scopol - calization of these 2 proteins, suggesting that VGF might exert amine, i.p.); (2) mice treated with saline (i.p.) plus scopolamine its function with the same process as BDNF in the prefrontal (0.025 mg/kg, i.p.); (3) mice treated with saline (i.p.) plus scopol - cortex and hippocampus. Our fluorescence immunohisto - amine (1 mg/kg, i.p.); (4) mice treated with saline (i.p.) plus scopol - chemistry showed that low doses of scopolamine (0.025 mg/kg) amine (3 mg/kg, i.p.); (5) mice treated with verapamil (5 mg/kg, i.p.) significantly increased the expression of VGF [F (2, 12) = 203.8, plus scopolamine (0.025 mg/kg, i.p.); (6) mice treated with vera - P < .01, Figure 4B; F (2, 12) = 48.34, P < .01, Figure 5B] and BDNF [F pamil (5 mg/kg, i.p.) plus scopolamine (1 mg/kg, i.p.); (7) mice (2, 12) = 113.2, P < .01, Figure 4C; F (2, 12) = 26.59, P < .01, Figure 5C] treated with verapamil (5 mg/kg, i.p.) plus scopolamine (3 mg/ in the hippocampal CA3 region and prefrontal cortex, respec - kg, i.p.). As described earlier, mice received i.p. administration tively. However, the higher dose of scopolamine (1 mg/kg) sig - of verapamil (5 mg/kg) 60 minutes before scopolamine, and the nificantly decreased the expression of VGF (Figure 4B and 5B) OFT followed with NORT were performed 24 hours after the and BDNF (Figure 4C and 5C) in the hippocampal CA3 and pre - last treatment. We did not observe significant changes in line frontal cortex regions. Figure 4. Effects of various doses of scopolamine on VGF and brain derived neurotrophic factor (BDNF) immunoreactivity and colocalization in the hippocampal CA3 regions of mice. (A) VGF and BDNF protein expression was examined using immunofluorescence of frozen mice hippocampal sections. Anti-VGF was labeled with an Alex Fluor 594 conjugated secondary antibody (red). Anti-BDNF antibody was labeled with an Alex Fluor 488 conjugated secondary antibody (green). The densities of both VGF and BDNF were significantly increased by low doses of scopolamine (0.025 mg/kg) and were significantly decreased by a high dose of scopolamine (1 mg/kg). The cellular localization of VGF and BDNF after scopolamine treatment was characterized by colocalization studies with markers of neurons (D = API). 5/gr n oup. **P < .01 compared with the control (saline plus saline) group. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/175/4093880 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Yu et al. | 181 crossing [F (6, 56) = 1.623, P = 1.623; Figure 6A] or rearings [F (6, the immobility time compared with those receiving saline plus 56) = 0.6704, P = .6739; Figure 6B]. However, the recognition index saline (1 mg/kg, P < .05 and 3 mg/kg, P < .01) (Figure 6D). However, in NORT exhibited a significant reduction in mice treated with verapamil did not modulate the immobility time alterations high doses of scopolamine [F (6, 56) = 11.17, 1 mg/kg, P < .01 and associated with scopolamine treatment (data not shown). 3 mg/kg, P < .01] (Figure 6C) compared with those treated with saline plus saline. However, treatment with verapamil plus sco - Inhibition of L-VDCC Prevents the Upregulation of polamine (1 or 3 mg/kg) did not affect the recognition deficits VGF and BDNF by Scopolamine at Low Doses in Mice in mice induced by high doses of scopolamine compared with BDNF and VGF were found to play critical roles in rapid-acting that of the saline plus scopolamine (1 or 3 mg/kg) groups. By further examination of the depression-like behaviors in FST, we antidepressant-like actions of certain drugs ( Li et al. 2010 ; Voleti et al., 2013 ; Lepack et al. 2014 ; Lu et al., 2014 ; Pazini et al., 2016 ; confirmed that a low dose (0.025 mg/kg) of scopolamine [F (6, 56) = 11.65, P < .05] (Figure 6D) significantly decreased the immo - Girgenti et al., 2017 ). To clarify whether BDNF and VGF activ -a tion also facilitates the mechanistic action of scopolamine and bility time compared with the saline plus saline treatment, sug - gesting a potent antidepressant-like effect of scopolamine at whether L-VDCC participated in this process, we performed a western-blot analysis of BDNF and VGF in select murine brain this concentration in mice. Intriguingly, pretreatment with v- era pamil before scopolamine (0.025 mg/kg) injection significantly regions (prefrontal cortex and hippocampus) 4 days after treat - ment with scopolamine (0.025, 0.05, 0.1, 1, and 3 mg/kg, i.p.) with blocked the aforementioned antidepressant-like effects ( P < .05; Figure 6D). Additionally, mice treated with high doses of scopola - and without verapamil (5 mg/kg). The 1-way ANOVA of BDNF [hippocampus, F (6, 14) = 38.45, P < .01, Figure 7B; prefrontal cortex, mine (1 and 3 mg/kg) consistently showed significant increase in Figure 5. Effects of various doses of scopolamine on VGF and brain derived neurotrophic factor (BDNF) immunoreactivity and colocalization in the prefrontal cortex of mice. (A) VGF and BDNF protein expression was examined using immunofluorescence of frozen mice prefrontal cortex sections. Anti-VGF was labeled with an Alex Fluor 594 conjugated secondary antibody (red). Anti-BDNF antibody was labeled with an Alex Fluor 488 conjugated secondary antibody (green). The densities of both VGF and BDNF were significantly increased by a low dose of scopolamine (0.025 mg/kg) and were significantly decreased by a high dose of scopolamine (1 mg/kg). The cellular localization of VGF and BDNF after scopolamine treatment was characterized by colocalization studies with markers of neurons (D = API). 5/gr n oup. P < .05, ** P < .01 compared with the control (saline plus saline) group. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/175/4093880 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 182 | International Journal of Neuropsychopharmacology, 2018 Figure 6. L-type voltage-dependent calcium channel (L-VDCC) involved in the antidepressant-like effects of low-dose scopolamine in mice. Mice were pre-injected with verapamil (5 mg/kg, i.p.) followed by scopolamine (0.025, 1 and 3 mg/kg, i.p.), and the open field test (OFT) was conducted 24 hours after the last drug treatment. None of the treatments affected locomotor activity, reflected by the line crossings (A) and rearings (B) in the mice. (C) The novel object recognition index was examined in the novel object recognition test (NORT). (D) Immobility time of mice was measured in the forced swimming test (FST). Pretreatment with verapamil significantly reversed the antidepressant-like effects of scopolamine (0.025 mg/kg). The data are expressed as the mean ± SEM (n = 9/group). ** P < .05 and P < .01, compared with saline plus saline group; P < .05, compared with saline plus scopolamine (0.025 mg/kg) group. F (6, 14) = 18.45, P < .01, Figure 7E] and VGF [hippocampus, F (6, 2017; Newhouse and Dumas, 2015 ), leading to the so-called 14) = 21.62, P < .01, Figure 7C; prefrontal cortex, F (6, 14) = 17.25, “cholinergic hypothesis of Alzheimer’s disease (AD)” ( Francis P < .01, Figure 7F] data showed significant changes in any test et al., 1999). The inhibitors of acetylcholinesterase, which group for any brain region. Specifically, a low dose of scopolamine works to increase acetylcholine (ACh) levels by inhibiting ACh (0.025 mg/kg) significantly increased levels of BDNF [hippocam- degradation, have therefore been used clinically as first-line pus, P < .01; prefrontal cortex, P < .05] and VGF [hippocampus, therapy in memory dysfunction (Lleó et al., 2006). Recently, P < .01; prefrontal cortex, P < .05] proteins in the hippocampus and scopolamine, a nonselective muscarinic receptor antagonist prefrontal cortex compared with the saline plus saline treatment. that is used to induce memory impairment in rodents (Pitsikas On the contrary, the high doses of scopolamine (1 and 3 mg/kg) and Gravanis, 2017; Hwang et al., 2017 ) based on the choliner - significantly decreased the BDNF [1 mg/kg, hippocampus, P < .01; gic hypothesis, was generated ( Francis et al., 1999 ; Klinkenberg prefrontal cortex, P < .05; 3 mg/kg, both P < .01] and VGF [all P < .05] and Blokland 2010 ) and used as a standard-reference drug for levels in all brain regions of the mice compared with the saline inducing age- and dementia-related cognitive deficits in the plus saline treatment. Notably, verapamil injection prevented the animals. Interestingly, the cholinergic system has been cur - induction of brain BDNF and VGF expression by low-dose sco - rently under investigation as a target for rapid-acting antide - polamine (0.025 mg/kg), as the BDNF [hippocampus, both P < .01; pressant interventions ( Drevets et al., 2013 ), with the evidence prefrontal cortex, both P < .01] and VGF levels in the hippocampus suggesting that hyper-sensitivity of the cholinergic system and prefrontal cortex of mice treated with verapamil plus sco - plays a role in the pathophysiology of depression Jano ( wsky polamine were significantly lower than those of the saline plus et al., 1972). Several randomized, double-blind, placebo-con - scopolamine (0.025 mg/kg)-treated mice. trolled studies have been conducted with i.v. injection of the anticholinergic agent scopolamine as an adjunctive or mono - therapy in subjects with depressive disorders ( Khajavi et al., 2012; Yang and Hashimoto, 2015; Drevets et al., 2013 ). However, Discussion the undesirable effects induced by scopolamine, especially Deficits in the cholinergic system in the brain have been asso- cognitive deficits ( Klinkenberg and Blokland, 2010 ), have par- ciated with dysfunction of cognitive ability ( Blake and Boccia, tially limited the use of this drug in psychiatric disorders. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/175/4093880 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Yu et al. | 183 Figure 7. Pretreatment with verapamil blocked the upregulation of scopolamine (0.025 mg/kg) on brain derived neurotrophic factor (BDNF) and VGF in the brains of mice. (A) and (D) representative immunoblots of BDNF and VGF detected by western blotting with tissues from the hippocampus (A) and prefrontal cortex (D); the remaining panels are the quantification of the immunoblotting bands of BDNF (B,E) and VGF (C,F). The data are expressed as the mean ± SEM (n = 3/group). * P < .05, ## **P < .01, compared with saline plus saline group; P < .01, compared with verapamil plus scopolamine (0.025 mg/kg) group. Preclinical models suggest that these memory deficits and It has been well demonstrated that high doses of scopola - rapid antidepressant-like effects can be recapitulated with a mine produce cognitive deficits in various behavioral tests in blockade of M-type muscarinic acetylcholine receptors and animal models (Klinkenberg and Blokland, 2010 ). In the current downregulation of BDNF and VGF in the brain Ghumatkar ( study, scopolamine (1 and 3 mg/kg, i.p.) significantly decreased et al., 2015 ; Jeon et al., 2017 ; Zhou et al., 2017); however, the the recognition index in the NORT and increased the immobil - underlying cellular mechanisms of BDNF and VGF changes and ity time in the FST, with no changes in the total number of line behavioral responses to various doses of scopolamine have not crossings and rearings, suggesting that high doses of scopola - been determined. Herein, we report different regulatory effects mine (1 and 3 mg/lg, i.p.) impair memory and induce depres - on memory and depression-like behaviors as well as changes sion-like behaviors without affecting locomotion. Our results in BDNF and VGF produced by various doses of scopolamine were consistent with previous reports in general, in which high treatment in mice. doses of scopolamine impair cognitive ability in rodents with Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/175/4093880 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 184 | International Journal of Neuropsychopharmacology, 2018 or without increased locomotor activity ( Hiramatsu and Inoue, phosphorylation levels in the hippocampus and cortex ( Chen 2000; Kwon et al., 2009 ; Jeon et al., 2017). It is important to note et al., 2016 ; Park et al., 2016 ), which may explain the downregu - that our results showed that lower doses of scopolamine at lation mechanisms of BDNF and VGF expressions at high doses 25 and 50 μg/kg (i.p.) produced significant antidepressant-like of scopolamine. effects, which is consistent with previous studies ( Wohleb et al., The activation of synaptic plasticity has also been proposed 2016) and contrast to the high-dose studies that typically use as an underlying biological effect for scopolamine’s rapid-acting 1- to 3-mg/kg doses of scopolamine to produce psychiatric dis - antidepressant-like action at a low dose. Similar to ketamine, orders (Almasi-Nasrabadi et al., 2012 ; Busquet et al., 2012 ; Lin scopolamine treatment acutely enhances extracellular glutam - et al., 2016 ). ate production in the striatum of rodents ( Rawls and McGinty, Given the involvement of BDNF and VGF in the rapid-acting 1998), which may activate synaptic plasticity pathways. antidepressant-like and memory-enhancing effects ( Lu et al., Increased glutamate release activates α-amino-3-hydroxy-5- 2014; Ma et al., 2016 ; Yang et al., 2016 ), it is likely that BDNF and methyl-4-isoxazolepropionic acid (AMPA) receptor, resulting VGF may also play a key role in the actions of various doses of in depolarization of the cell and an influx of calcium through scopolamine in mice. Notably, BDNF is a member of the neu - L-VDCC in the rapid-acting antidepressant-like actions of keta - rotrophin family and plays a critical role in the cognitive pr - o mine ( Lepack et al., 2014 ; Chowdhury et al., 2017 ; Wohleb et al., cesses (de Assis and de Almondes, 2017;) and depression-like 2017). Interestingly, scopolamine’s antidepressant-like effects behaviors (Jiang et al., 2017 ). Additionally, BDNF plays a critical were blocked by pretreatment with mammalian target of rapa - role in the synaptic activity activation directly or by exerting a mycin complex 1 and AMPA antagonists (Voleti et al., 2013 ), positive feedback on synaptic activity ( Arancio and Chao, 2007). similar to ketamine (Li et al., 2010). As noted above, scopola - Recently, previous studies reported a marked downregulation mine’s mechanism of action goes beyond simple M-type mus - of the BDNF-mediated signaling in the prefrontal cortex and carinic acetylcholine receptor antagonism or even activation of hippocampus of various depression and memory deficit mod - the AMPA receptor. Importantly, L-VDCC may play crucial roles els (Zhou et al., 2017 ; Xiang et al., 2017 ). However, upregulation in the actions of scopolamine. To further validate the speci - of BDNF promoted rapid and sustained antidepressant-like ficity of L-VDCC-induced regulation of BDNF and VGF expres- and memory-enhancing effects, thus implicating the BDNF- sion after various doses of scopolamine treatment in mice, the mediated signaling in the prefrontal cortex and hippocampus, L-VDCC blocker verapamil (5 mg/kg, i.p.) was administered as which plays an important role in regulating the effects of v -ari a pretreatment before scopolamine. When effective doses of ous doses of scopolamine. Herein, we confirmed that low doses scopolamine (0.025, 1 and 3 mg/kg, i.p.) were administered with of scopolamine (25 and 50 μg/kg) significantly increased BDNF the verapamil, the low dose of scopolamine (0.025 mg/kg) plus levels in the prefrontal cortex and hippocampus in addition to verapamil showed significant memory deficits and depression- rapid-acting antidepressant-like action in mice. Growing e -vi like behaviors in the NORT and FST compared with the scopol - dence suggests that the antidepressant-like effects of ketamine amine (0.025 mg/kg) single treatment. Specifically, the low dose and scopolamine in rodent models are caused by an influx of of scopolamine (0.025 mg/kg) produced increases in BDNF and extracellular glutamate, elevated BDNF, and activation of the VGF, which were completely blocked by verapamil pretreatment. BDNF/tropomyosin-related kinase receptor B signaling ( Lepack These findings demonstrate a requirement of L-VDCC in the et al., 2014 ; Wohleb et al., 2016 , 2017), which may explain the rapid-acting antidepressant-like effects of low doses of scopol - BDNF upregulation by low doses of scopolamine in our current amine in mice. work. Recently, VGF, a secreted neuropeptide was found to be downregulated in the brain of animal models of depression Conclusions (Hunsberger et al., 2007 ; Thakker-Varia et al., 2007 ; Lin et al., 2014). Additionally, VGF involves in the antidepressant-like Taken together, our findings suggest that the different changes effects dependent on the BDNF/TrkB/CREB signaling in mice of behaviors produced by different doses of scopolamine are (Hunsberger et al., 2007 ; Thakker-Varia et al., 2007 ; Lin et al., dependent on the opposing regulation of BDNF and VGF levels 2014; Lu et al., 2014). Consistent with our expectations in our in the hippocampus and prefrontal cortex of mice. Additionally, current work, the levels of VGF were significantly increased in our results further implicate that L-VDCC in the hippocampus the hippocampus and prefrontal cortex by low doses of scopol - and prefrontal cortex may be involved in the behavioral and amine, indicating the similar manner with the BDNF changes molecular responses to scopolamine. regulated by low doses of scopolamine in mice. In contrast to the effects of low doses of scopolamine, our Acknowledgments data showed that the high doses of scopolamine (1 and 3 mg/kg) significantly produced the memory deficits and depression- This research was supported by the National Natural Science like behaviors in mice. Our present data are consistent with Foundation of China (No. 81671337; No. 81201050; No. 81541087); the previous studies that demonstrated that the high doses of sco - Natural Science Foundation of Zhejiang Province (No. LQ12H09001), polamine induced memory impairments and anxiety-like and Natural Science Foundation of Ningbo (No. 2012A610251; No. depression-like behaviors in rats ( Aydin et al., 2016 ; Bagci et al., 2017A610212), and the Ningbo municipal innovation team of life 2016). Additionally, our biochemical studies found that the le - v science and health (2015C110026). This project is also sponsored by els of BDNF and VGF in the hippocampus and prefrontal cortex K.C. Wong Magna funded in Ningbo University and the Li Dak Sum were significantly decreased after high doses of scopolamine (1 YiP Yio Chin Kenneth Li Marine Biopharmaceutical Development and 3 mg/kg) treatment, which may explain the depression-like Fund, National 111 Project of China. behaviors and memory deficits in mice. Previous studies have revealed that scopolamine (1 or 3 mg/kg) induced an increase in acetylcholinesterase activity as well as decreases in BDNF Statement of Interest protein expression and cAMP response element-binding pro - tein, extracellular regulated kinase 1/2, and protein kinase B None. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/175/4093880 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Yu et al. | 185 Hiramatsu M, Inoue K (2000) Improvement by low doses of noci- References ceptin on scopolamine-induced impairment of learning and/ Almasi-Nasrabadi M, Javadi-Paydar M, Mahdavian S, Babaei R, or memory. 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International Journal of Neuropsychopharmacology – Oxford University Press
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