Neuroscience Letters 599 (2015) 69–74
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Antidepressant action via the nitric oxide system: A pilot study in an acute depressive model induced by arginin. Yuta Yoshino ∗ , Shinichiro Ochi, Kiyohiro Yamazaki, Shunsuke Nakata, Masao Abe, Yoko Mori, Shu-ichi Ueno Department of Neuropsychiatry, Molecules and Function, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan
h i g h l i g h t s • • • •
iNOS expression in the brain is increased with arginine depressive rat model. Milnacipran elevates serum NO in this model. Fluoxetine raises brain eNOS expression in this model. Either milnacipran or mirtazapine do not change NOS expressions in this model.
a r t i c l e
i n f o
Article history: Received 4 February 2015 Received in revised form 18 May 2015 Accepted 21 May 2015 Available online 23 May 2015 Keywords: Depression Antidepressant Rat Nitric oxide Nitric oxide synthase
a b s t r a c t Nitric oxide (NO) may be a neurotransmitter related to major depressive disorder (MDD) because the selective neuronal NO synthase (NOS) inhibitor, 7-nitroindazole, induces dose-dependent antidepressant-like effects. However, its role in MDD is not yet known. The purpose of our study was to determine if antidepressants improve depression via the NO pathway using an acute depressive rat model induced by l-arginine (AR). Three types of antidepressants were examined, fluoxetine (FLX, 10 mg/kg), milnacipran (MIL, 30 mg/kg), and mirtazapine (MIR, 10 mg/kg), in a depressive model that used AR (750 mg/kg) pretreatment. mRNA expression levels of three NOS subtypes were analyzed by real-time PCR, as well as serum NO levels. Significant increases in iNOS mRNA expression levels were found in brain regions after AR treatment, although the eNOS gene tended to decrease with AR injection. After antidepressant treatment, there were no mRNA expression changes in either nNOS or iNOS. However, eNOS mRNA expression significantly increased with FLX (cerebellum, P = 0.011; hippocampus, P = 0.011; midbrain, P = 0.011; pons, P = 0.013; striatum, P = 0.011; and thalamus, P < 0.001). There was a statistically significant increase in serum NO levels with MIL treatment (P = 0.011). We conclude that changes in eNOS mRNA levels in the brain with FLX treatment, and amount of serum NO with MIL treatment may be related to antidepressant effects of both agents, but further experiments are needed to confirm involvement of the NO system in MDD. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Major depressive disorder (MDD) is an important mental disorder, with a lifetime prevalence rate of 10–30% for women and 7–15% for men [1]. Changes in monoamine neurotransmitters in the brain, including norepinephrine, serotonin, and dopamine, are implicated in MDD pathogenesis [2]. Several studies on the nitric oxide (NO) system suggest that it may also be involved in MDD. NO levels in MDD patients have been found to be both increased
∗ Corresponding author. Tel.: +81 89 960 5315; fax: +81 89 960 5317. E-mail address: [email protected] (Y. Yoshino). http://dx.doi.org/10.1016/j.neulet.2015.05.043 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
[3] and decreased [4], potentially due to differing NO subtypes, different states, or different treatments received. In particular, MDD patients that attempt suicide are reported to have high plasma NO levels [5]. NO is synthesized from l-arginine (AR) by three NO synthase (NOS) isoforms: neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS) [6]. The non-selective NOS inhibitor, l-NG -nitroarginine methyl ester (l-NAME), and the selective neuronal NOS inhibitor, 7-nitroindazole, induce dose-dependent antidepressant-like effects in the forced swimming test [7–9]. Furthermore, hippocampal NOS expression is significantly increased in depressed patients [10]. Thus, we propose that antidepressants may exert an effect via the brain NO system. To our knowledge,
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Fig. 1. mRNA expression of NOS subtypes. (A) nNOS, (B) iNOS, and (C) eNOS in brain tissue with or without AR treatment. Values are expressed as mean + S.E.M. (n = 6; *P < 0.05). NS, normal saline; AR, l-arginine; NOS, nitric oxide synthase; nNOS, neuronal NOS; iNOS, inducible NOS; eNOS, endothelial NOS; cer, cerebellum; fro, frontal cortex; hip, hippocampus; mid, midbrain; olf, olfactory; str, striatum; tem, temporal cortex; tha, thalamus.
there are only a few reports investigating antidepressant mechanisms of the NO system. Here, we determine if antidepressants representing distinct drug categories, specifically, fluoxetine as a selective serotonin reuptake inhibitor (SSRI), milnacipran as a sero-
tonin noradrenalin reuptake inhibitor (SNRI), and mirtazapine as a noradrenalin serotonin system antidepressant (NaSSA), also work as NO modulators in an acute depressive model induced by AR. Our findings will provide new information on MDD treatment options.
Y. Yoshino et al. / Neuroscience Letters 599 (2015) 69–74
2. Materials and methods 2.1. Animals Male adult Wistar rats weighing 180–200 g were purchased from CLEA Japan, Inc. (Tokyo, Japan). Rats were housed three per cage (temperature, 22 ± 2 ◦ C), with free access to food and water and a 12 h light/12 h dark cycle (lights on at 06:00 h). All experiments were conducted in accordance with the Guidelines for Animal Experimentation of Ehime University Graduate School of Medicine (Ehime, Japan).
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the Shapiro–Wilk test. Comparisons of gene expression levels of the three NOS isoforms and serum NO levels between rats treated with or without AR were determined by Student t tests if the data were normally distributed, or Mann–Whitney U tests if the data were not normally distributed. In AR groups treated with FLX, MIL, and MIR, gene expression levels of the three NOS isoforms and serum NO levels were compared by one-way analyses of variance (ANOVA) with post hoc Dunnett’s t tests and Dunnett’s T3 tests if the data were normally distributed, or Kruskal–Wallis tests with post hoc steel tests (EZR version 1.26; [20]) if the data were not normally distributed. Statistical significance was defined at the 95% level (P value = 0.05).
2.2. Drugs The following drugs were used: fluoxetine hydrochloride (FLX), milnacipran hydrochloride (MIL), and l-arginine (AR) (all Wako Pure Chemical Industries, Osaka, Japan), and mirtazapine (MIR) (MSD, Tokyo, Japan). FLX, MIL, and AR were dissolved in 0.9% saline (normal saline, NS). MIR was dissolved in 0.1 N HCl and the pH of the final solution adjusted to 7.0 with phosphate buffer at 37 ◦ C. The injected drug doses were: FLX, 10 mg/kg [11]; MIL, 30 mg/kg [12]; MIR, 10 mg/kg [13]; and AR, 750 mg/kg [14]. Each drug dosage was reported to affect behavioral and/or biochemical status in past studies. 2.3. Serum NO measurement Serum NO was measured by the Griess reaction method [15] modified by Tracey et al. [16], using a commercially available kit (Funakoshi, Tokyo, Japan). 2.4. Experimental procedure High dose (1000 mg/kg) AR causes a depressive state in animals, which is inhibited by NOS inhibitors [17]. Treatment with AR is a popular method to induce depressive rodent models [14]. Rats were peritoneally treated with AR (750 mg/kg) or NS, and after 30 min, each agent or NS was injected. An hour later, all rats were sacrificed by decapitation and brain tissue from nine regions (frontal cortex, temporal cortex, striatum, thalamus, hippocampus, midbrain, pons, cerebellum, and olfactory bulb) bilaterally dissected on an ice-cold stage according to Glowinski and Iversen [18]. Fresh tissues were weighed and stored at −80 ◦ C before use. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research [19] was followed. 2.5. Total RNA isolation and real-time PCR method for gene expression RNA was extracted from each brain tissue using the RNeasy kit, including RNase-Free DNase treatment (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. RNA quantity was validated using the Nanodrop1000 (Thermo Scientific, Waltham, MA, USA). RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Comparative evaluation of quantitative real-time PCR was performed using TaqMan probes (Assay ID: nNOS, Rn00583793 m1; iNOS, Rn00561646 m; and eNOS, Rn02132634 s1; Applied Biosystems), with the TaqMan gene expression master mix and a StepOnePlus real-time PCR system (Applied Biosystems). GAPDH was used as an internal standard. 2.6. Statistical analysis Statistical analyses was performed using SPSS 22.0 software (IBM Japan, Tokyo, Japan). Normality tests were performed using
3. Results 3.1. NOS gene expression changes induced by AR There were no differences for either nNOS (Fig. 1A) or eNOS (Fig. 1C). However, mRNA expression of iNOS with AR treatment was significantly higher than NS in several brain regions: cerebellum, P = 0.002; frontal cortex, P = 0.045; midbrain, P = 0.037; olfactory bulb, P = 0.049; pons, P = 0.006; striatum, P = 0.021; temporal cortex, P = 0.002; and thalamus, P = 0.002 (Fig. 1B). 3.2. NOS gene expression changes with AR and antidepressant treatment There were no mRNA expression changes for nNOS (Fig. 2A) or iNOS (Fig. 2B) with antidepressant treatment. However, eNOS mRNA expression levels with FLX treatment were significantly higher than without FLX in several regions: cerebellum, P = 0.011; hippocampus, P = 0.011; midbrain, P = 0.011; pons, P = 0.013; striatum, P = 0.011; and thalamus, P < 0.001 (Fig. 2C). 3.3. Serum NO levels NO levels after AR treatment were higher than the NS group, but not statistically significant (P = 0.115, Fig. 3A). NO levels with MIL treatment were significantly higher than without MIL (P = 0.011, Fig. 3B), but were not changed with the other antidepressants. 4. Discussion Here, we show three important results using an acute depressive model induced by AR injection. First, we found significantly increases iNOS mRNA expression levels in several brain regions after AR treatment, whereas eNOS tended to decrease with AR injection. Increased iNOS expression in this model may be due to immune reactions because iNOS is involved in autoimmune reactions [21]. Altered iNOS expression due to AR injection may represent a pathological change associated with depression because a link has been established between neurotransmitter dysfunction and immune activation in MDD [22]. Gao et al. [23] observed low activities of both nNOS and eNOS in the hypothalamus in a chronic depression rat model. However, we did not find changes in either NOS subtype. Second, we found that FLX significantly increased eNOS mRNA expression in several brain regions, although the other antidepressants (MIL and MIR) did not change mRNA expression of any NOS subtype in this acute depressive model (Fig. 3). There are several studies on vascular changes in depression, for example, ischemic lesions located in the dorsolateral prefrontal cortex were observed in depressed subjects [24], and endothelial changes in MDD patients [25]. eNOS regulates blood vessel constriction and blood pressure [26]. Chrapko et al. [27] reported that platelet eNOS
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Fig. 2. mRNA expression of NOS subtypes. (A) nNOS, (B) iNOS, and (C) eNOS in brain tissue treated with FLX, MIL, and MIR after AR injection. Values are expressed as mean + S.E.M. (n = 6; *P < 0.05, **P < 0.001). AR, l-arginine; NOS, nitric oxide synthase; nNOS, neuronal NOS; iNOS, inducible NOS; eNOS, endothelial NOS; FLX, fluoxetine; MIL, milnacipran hydrochloride; MIR, mirtazapine; cer, cerebellum; fro, frontal cortex; hip, hippocampus; mid, midbrain; olf, olfactory; str, striatum; tem, temporal cortex; tha, thalamus.
activity and plasma NO levels are decreased in MDD patients. Additionally, eNOS has been reported to regulate BDNF expression and influence progenitor cell proliferation, neuronal migration,
and neurite outgrowth [28]. Malberg et al. [29] reported that chronic antidepressant treatment increases hippocampal neurogenesis. Here, eNOS mRNA expression levels tended to decrease
Y. Yoshino et al. / Neuroscience Letters 599 (2015) 69–74
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Acknowledgments We wish to thank Ms. Takako Muneta for her technical assistance. This work was partially supported by a Health and Labor Science Research Grant from the Japanese Ministry of Health, Labour and Welfare, a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and the Ehime Graduate University of Medicine’s Good Practice Fund.
References
Fig. 3. NO levels. (A) Comparison between NS and AR groups, and (B) AR controls compared with +FLX, +MIL, and +MIR. Values are expressed as mean + S.E.M. (n = 6; *P < 0.05). NO, nitric oxide; NS, normal saline; AR, l-arginine; FLX, fluoxetine; MIL, milnacipran hydrochloride; MIR, mirtazapine.
in depressed model rats induced by AR treatment, and significantly increased with FLX treatment, thus this change may relate to its antidepressant effect. However, we detected no changes with MIL or MIR. Finally, NO amount significantly increased with MIL treatment (P = 0.011). In chronic depression rat models, high NO levels are observed with antidepressants [23]. Paroxetine was also reported to raise serum NO in MDD patients [30], and MDD patients have low serum NO compared with control subjects, which increases after MIL treatment [31]. In our model, increased NO due to MIL may be related to its antidepressant effect, although we observed no increases with the other antidepressants. Nevertheless, NO levels in MDD patients are reported to both increase [3], and decrease [4]. Further studies are needed to determine if MDD pathogenesis and antidepressants affect serum NO levels. Our study has several limitations. First, we investigated only the NO system in our acute depressive model. Nevertheless, changes in NOS expression and biological effects with some antidepressants were shown, even in this acute model. Comparing effective antidepressant expression changes in the NO system to investigate chronic depressive models with subacute antidepressant treatment will be performed in the future. Second, the sample size (n = 6, each group) is small, although we believe it is big enough to conclude our results, with taking animal welfare into consideration.
Conflict of interest None to declare.
[1] J.A. Blendy, The role of CREB in depression and antidepressant treatment, Biol. Psychiatry 59 (12) (2006) 1144–1150. [2] K. Chopra, B. Kumar, A. Kuhad, Pathobiological targets of depression, Expert Opin. Ther. Targets 15 (4) (2011) 379–400. [3] M. Talarowska, P. Gałecki, M. Maes, A. Orzechowska, M. Chamielec, G. Bartosz, E. Kowalczyk, Nitric oxide plasma concentration associated with cognitive impairment in patients with recurrent depressive disorder, Neurosci. Lett. 510 (2) (2012) 127–131. [4] M.L. Selley, Increased (E)-4-hydroxy-2-nonenal and asymmetric dimethylarginine concentrations and decreased nitric oxide concentrations in the plasma of patients with major depression, J. Affect. Disord. 80 (2–3) (2004) 249–256. [5] B.H. Lee, S.W. Lee, D. Yoon, H.J. Lee, J.C. Yang, S.H. Shim, D.H. Kim, S.H. Ryu, C. Han, Y.K. Kim, Increased plasma nitric oxide metabolites in suicide attempters, Neuropsychobiology 53 (3) (2006) 127–132. ˜ [6] A.M. Magarinos, B.S. McEwen, Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: involvement of glucocorticoid secretion and excitatory amino acid receptors, Neuroscience 69 (1) (1995) 89–98. [7] F. Yildiz, B.F. Erden, G. Ulak, T. Utkan, N. Gacar, Antidepressant-like effect of 7-nitroindazole in the forced swimming test in rats, Psychopharmacology (Berl.) 149 (1) (2000) 41–44. [8] A. Spiacci Jr, F. Kanamaru, F.S. Guimarães, R.M. Oliveira, Nitric oxide-mediated anxiolytic-like and antidepressant-like effects in animal models of anxiety and depression, Pharmacol. Biochem. Behav. 88 (3) (2008) 247–255. [9] F.R. Ferreira, A.M. Oliveira, A.R. Dinarte, D.G. Pinheiro, L.J. Greene, W.A. Silva Jr, S.R. Joca, F.S. Guimarães, Changes in hippocampal gene expression by 7-nitroindazole in rats submitted to forced swimming stress, Genes Brain Behav. 11 (3) (2012) 303–313. [10] R.M. Oliveira, F.S. Guimarães, J.F. Deakin, Expression of neuronal nitric oxide synthase in the hippocampal formation in affective disorders, Braz. J. Med. Biol. Res. 41 (4) (2008) 333–341. [11] S. Lapmanee, J. Charoenphandhu, N. Charoenphandhu, Beneficial effects of fluoxetine, reboxetine, venlafaxine, and voluntary running exercise in stressed male rats with anxiety- and depression-like behaviors, Behav. Brain Res. 250 (2013) 316–325. [12] K.J. Naegeli, J.A. O’Connor, P. Banerjee, D.A. Morilak, Effects of milnacipran on cognitive flexibility following chronic stress in rats, Eur. J. Pharmacol. 703 (1–3) (2013) 62–66. [13] S. Yamamura, M. Abe, M. Nakagawa, S. Ochi, S. Ueno, M. Okada, Different actions for acute and chronic administration of mirtazapine on serotonergic transmission associated with raphe nuclei and their innervation cortical regions, Neuropharmacology 60 (4) (2011) 550–560. [14] G.F. Zhang, N. Wang, J.Y. Shi, S.X. Xu, X.M. Li, M.H. Ji, Z.Y. Zuo, Z.Q. Zhou, J.J. Yang, Inhibition of the l-arginine-nitric oxide pathway mediates the antidepressant effects of ketamine in rats in the forced swimming test, Pharmacol. Biochem. Behav. 110 (2013) 8–12. [15] Griess, P.H.H. Bemerkungen zu der abhandlung der Weselsky und Benedikt, Ueber einige azoverbindungen, Chemische Berichte 12 (1879) 426–428. [16] W.R. Tracey, J. Tse, G. Carter, Lipopolysaccharide-induced changes in plasma nitrite and nitrate concentrations in rats and mice: pharmacological evaluation of nitric oxide synthase inhibitors, J. Pharmacol. Exp. Ther. 272 (1995) 1011–1015. [17] Y. Ergün, U.G. Ergün, Prevention of pro-depressant effect of l-arginine in the forced swim test by NG-nitro-l-arginine and [1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one], Eur. J. Pharmacol. 554 (2–3) (2007) 150–154. [18] J. Glowinski, L.L. Iversen, Regional studies of catecholamines in the rat brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]dopa in various regions of the brain, J. Neurochem. 13 (8) (1996) 655–669. [19] National Research Council (US) Committee on Guidelines for the Use of Animals in Neuroscience and Behavioral Research, Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research, National Academies Press (US), Washington (DC), 2003. [20] Y. Kanda, Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics, Bone Marrow Transplant. 48 (3) (2013) 452–458. [21] H. Koprowski, Y.M. Zheng, E. Heber-Katz, N. Fraser, L. Rorke, Z.F. Fu, C. Hanlon, B. Dietzschold, In vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases, Proc. Natl. Acad. Sci. U. S. A. 90 (7) (1993) 3024–3027.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Antidepressant action via the nitric oxide system: A pilot study in an acute depressive model induced by arginin. Yuta Yoshino ∗ , Shinichiro Ochi, Kiyohiro Yamazaki, Shunsuke Nakata, Masao Abe, Yoko Mori, Shu-ichi Ueno Department of Neuropsychiatry, Molecules and Function, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan
h i g h l i g h t s • • • •
iNOS expression in the brain is increased with arginine depressive rat model. Milnacipran elevates serum NO in this model. Fluoxetine raises brain eNOS expression in this model. Either milnacipran or mirtazapine do not change NOS expressions in this model.
a r t i c l e
i n f o
Article history: Received 4 February 2015 Received in revised form 18 May 2015 Accepted 21 May 2015 Available online 23 May 2015 Keywords: Depression Antidepressant Rat Nitric oxide Nitric oxide synthase
a b s t r a c t Nitric oxide (NO) may be a neurotransmitter related to major depressive disorder (MDD) because the selective neuronal NO synthase (NOS) inhibitor, 7-nitroindazole, induces dose-dependent antidepressant-like effects. However, its role in MDD is not yet known. The purpose of our study was to determine if antidepressants improve depression via the NO pathway using an acute depressive rat model induced by l-arginine (AR). Three types of antidepressants were examined, fluoxetine (FLX, 10 mg/kg), milnacipran (MIL, 30 mg/kg), and mirtazapine (MIR, 10 mg/kg), in a depressive model that used AR (750 mg/kg) pretreatment. mRNA expression levels of three NOS subtypes were analyzed by real-time PCR, as well as serum NO levels. Significant increases in iNOS mRNA expression levels were found in brain regions after AR treatment, although the eNOS gene tended to decrease with AR injection. After antidepressant treatment, there were no mRNA expression changes in either nNOS or iNOS. However, eNOS mRNA expression significantly increased with FLX (cerebellum, P = 0.011; hippocampus, P = 0.011; midbrain, P = 0.011; pons, P = 0.013; striatum, P = 0.011; and thalamus, P < 0.001). There was a statistically significant increase in serum NO levels with MIL treatment (P = 0.011). We conclude that changes in eNOS mRNA levels in the brain with FLX treatment, and amount of serum NO with MIL treatment may be related to antidepressant effects of both agents, but further experiments are needed to confirm involvement of the NO system in MDD. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Major depressive disorder (MDD) is an important mental disorder, with a lifetime prevalence rate of 10–30% for women and 7–15% for men [1]. Changes in monoamine neurotransmitters in the brain, including norepinephrine, serotonin, and dopamine, are implicated in MDD pathogenesis [2]. Several studies on the nitric oxide (NO) system suggest that it may also be involved in MDD. NO levels in MDD patients have been found to be both increased
∗ Corresponding author. Tel.: +81 89 960 5315; fax: +81 89 960 5317. E-mail address: [email protected] (Y. Yoshino). http://dx.doi.org/10.1016/j.neulet.2015.05.043 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
[3] and decreased [4], potentially due to differing NO subtypes, different states, or different treatments received. In particular, MDD patients that attempt suicide are reported to have high plasma NO levels [5]. NO is synthesized from l-arginine (AR) by three NO synthase (NOS) isoforms: neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS) [6]. The non-selective NOS inhibitor, l-NG -nitroarginine methyl ester (l-NAME), and the selective neuronal NOS inhibitor, 7-nitroindazole, induce dose-dependent antidepressant-like effects in the forced swimming test [7–9]. Furthermore, hippocampal NOS expression is significantly increased in depressed patients [10]. Thus, we propose that antidepressants may exert an effect via the brain NO system. To our knowledge,
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Y. Yoshino et al. / Neuroscience Letters 599 (2015) 69–74
Fig. 1. mRNA expression of NOS subtypes. (A) nNOS, (B) iNOS, and (C) eNOS in brain tissue with or without AR treatment. Values are expressed as mean + S.E.M. (n = 6; *P < 0.05). NS, normal saline; AR, l-arginine; NOS, nitric oxide synthase; nNOS, neuronal NOS; iNOS, inducible NOS; eNOS, endothelial NOS; cer, cerebellum; fro, frontal cortex; hip, hippocampus; mid, midbrain; olf, olfactory; str, striatum; tem, temporal cortex; tha, thalamus.
there are only a few reports investigating antidepressant mechanisms of the NO system. Here, we determine if antidepressants representing distinct drug categories, specifically, fluoxetine as a selective serotonin reuptake inhibitor (SSRI), milnacipran as a sero-
tonin noradrenalin reuptake inhibitor (SNRI), and mirtazapine as a noradrenalin serotonin system antidepressant (NaSSA), also work as NO modulators in an acute depressive model induced by AR. Our findings will provide new information on MDD treatment options.
Y. Yoshino et al. / Neuroscience Letters 599 (2015) 69–74
2. Materials and methods 2.1. Animals Male adult Wistar rats weighing 180–200 g were purchased from CLEA Japan, Inc. (Tokyo, Japan). Rats were housed three per cage (temperature, 22 ± 2 ◦ C), with free access to food and water and a 12 h light/12 h dark cycle (lights on at 06:00 h). All experiments were conducted in accordance with the Guidelines for Animal Experimentation of Ehime University Graduate School of Medicine (Ehime, Japan).
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the Shapiro–Wilk test. Comparisons of gene expression levels of the three NOS isoforms and serum NO levels between rats treated with or without AR were determined by Student t tests if the data were normally distributed, or Mann–Whitney U tests if the data were not normally distributed. In AR groups treated with FLX, MIL, and MIR, gene expression levels of the three NOS isoforms and serum NO levels were compared by one-way analyses of variance (ANOVA) with post hoc Dunnett’s t tests and Dunnett’s T3 tests if the data were normally distributed, or Kruskal–Wallis tests with post hoc steel tests (EZR version 1.26; [20]) if the data were not normally distributed. Statistical significance was defined at the 95% level (P value = 0.05).
2.2. Drugs The following drugs were used: fluoxetine hydrochloride (FLX), milnacipran hydrochloride (MIL), and l-arginine (AR) (all Wako Pure Chemical Industries, Osaka, Japan), and mirtazapine (MIR) (MSD, Tokyo, Japan). FLX, MIL, and AR were dissolved in 0.9% saline (normal saline, NS). MIR was dissolved in 0.1 N HCl and the pH of the final solution adjusted to 7.0 with phosphate buffer at 37 ◦ C. The injected drug doses were: FLX, 10 mg/kg [11]; MIL, 30 mg/kg [12]; MIR, 10 mg/kg [13]; and AR, 750 mg/kg [14]. Each drug dosage was reported to affect behavioral and/or biochemical status in past studies. 2.3. Serum NO measurement Serum NO was measured by the Griess reaction method [15] modified by Tracey et al. [16], using a commercially available kit (Funakoshi, Tokyo, Japan). 2.4. Experimental procedure High dose (1000 mg/kg) AR causes a depressive state in animals, which is inhibited by NOS inhibitors [17]. Treatment with AR is a popular method to induce depressive rodent models [14]. Rats were peritoneally treated with AR (750 mg/kg) or NS, and after 30 min, each agent or NS was injected. An hour later, all rats were sacrificed by decapitation and brain tissue from nine regions (frontal cortex, temporal cortex, striatum, thalamus, hippocampus, midbrain, pons, cerebellum, and olfactory bulb) bilaterally dissected on an ice-cold stage according to Glowinski and Iversen [18]. Fresh tissues were weighed and stored at −80 ◦ C before use. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research [19] was followed. 2.5. Total RNA isolation and real-time PCR method for gene expression RNA was extracted from each brain tissue using the RNeasy kit, including RNase-Free DNase treatment (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. RNA quantity was validated using the Nanodrop1000 (Thermo Scientific, Waltham, MA, USA). RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Comparative evaluation of quantitative real-time PCR was performed using TaqMan probes (Assay ID: nNOS, Rn00583793 m1; iNOS, Rn00561646 m; and eNOS, Rn02132634 s1; Applied Biosystems), with the TaqMan gene expression master mix and a StepOnePlus real-time PCR system (Applied Biosystems). GAPDH was used as an internal standard. 2.6. Statistical analysis Statistical analyses was performed using SPSS 22.0 software (IBM Japan, Tokyo, Japan). Normality tests were performed using
3. Results 3.1. NOS gene expression changes induced by AR There were no differences for either nNOS (Fig. 1A) or eNOS (Fig. 1C). However, mRNA expression of iNOS with AR treatment was significantly higher than NS in several brain regions: cerebellum, P = 0.002; frontal cortex, P = 0.045; midbrain, P = 0.037; olfactory bulb, P = 0.049; pons, P = 0.006; striatum, P = 0.021; temporal cortex, P = 0.002; and thalamus, P = 0.002 (Fig. 1B). 3.2. NOS gene expression changes with AR and antidepressant treatment There were no mRNA expression changes for nNOS (Fig. 2A) or iNOS (Fig. 2B) with antidepressant treatment. However, eNOS mRNA expression levels with FLX treatment were significantly higher than without FLX in several regions: cerebellum, P = 0.011; hippocampus, P = 0.011; midbrain, P = 0.011; pons, P = 0.013; striatum, P = 0.011; and thalamus, P < 0.001 (Fig. 2C). 3.3. Serum NO levels NO levels after AR treatment were higher than the NS group, but not statistically significant (P = 0.115, Fig. 3A). NO levels with MIL treatment were significantly higher than without MIL (P = 0.011, Fig. 3B), but were not changed with the other antidepressants. 4. Discussion Here, we show three important results using an acute depressive model induced by AR injection. First, we found significantly increases iNOS mRNA expression levels in several brain regions after AR treatment, whereas eNOS tended to decrease with AR injection. Increased iNOS expression in this model may be due to immune reactions because iNOS is involved in autoimmune reactions [21]. Altered iNOS expression due to AR injection may represent a pathological change associated with depression because a link has been established between neurotransmitter dysfunction and immune activation in MDD [22]. Gao et al. [23] observed low activities of both nNOS and eNOS in the hypothalamus in a chronic depression rat model. However, we did not find changes in either NOS subtype. Second, we found that FLX significantly increased eNOS mRNA expression in several brain regions, although the other antidepressants (MIL and MIR) did not change mRNA expression of any NOS subtype in this acute depressive model (Fig. 3). There are several studies on vascular changes in depression, for example, ischemic lesions located in the dorsolateral prefrontal cortex were observed in depressed subjects [24], and endothelial changes in MDD patients [25]. eNOS regulates blood vessel constriction and blood pressure [26]. Chrapko et al. [27] reported that platelet eNOS
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Fig. 2. mRNA expression of NOS subtypes. (A) nNOS, (B) iNOS, and (C) eNOS in brain tissue treated with FLX, MIL, and MIR after AR injection. Values are expressed as mean + S.E.M. (n = 6; *P < 0.05, **P < 0.001). AR, l-arginine; NOS, nitric oxide synthase; nNOS, neuronal NOS; iNOS, inducible NOS; eNOS, endothelial NOS; FLX, fluoxetine; MIL, milnacipran hydrochloride; MIR, mirtazapine; cer, cerebellum; fro, frontal cortex; hip, hippocampus; mid, midbrain; olf, olfactory; str, striatum; tem, temporal cortex; tha, thalamus.
activity and plasma NO levels are decreased in MDD patients. Additionally, eNOS has been reported to regulate BDNF expression and influence progenitor cell proliferation, neuronal migration,
and neurite outgrowth [28]. Malberg et al. [29] reported that chronic antidepressant treatment increases hippocampal neurogenesis. Here, eNOS mRNA expression levels tended to decrease
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Acknowledgments We wish to thank Ms. Takako Muneta for her technical assistance. This work was partially supported by a Health and Labor Science Research Grant from the Japanese Ministry of Health, Labour and Welfare, a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and the Ehime Graduate University of Medicine’s Good Practice Fund.
References
Fig. 3. NO levels. (A) Comparison between NS and AR groups, and (B) AR controls compared with +FLX, +MIL, and +MIR. Values are expressed as mean + S.E.M. (n = 6; *P < 0.05). NO, nitric oxide; NS, normal saline; AR, l-arginine; FLX, fluoxetine; MIL, milnacipran hydrochloride; MIR, mirtazapine.
in depressed model rats induced by AR treatment, and significantly increased with FLX treatment, thus this change may relate to its antidepressant effect. However, we detected no changes with MIL or MIR. Finally, NO amount significantly increased with MIL treatment (P = 0.011). In chronic depression rat models, high NO levels are observed with antidepressants [23]. Paroxetine was also reported to raise serum NO in MDD patients [30], and MDD patients have low serum NO compared with control subjects, which increases after MIL treatment [31]. In our model, increased NO due to MIL may be related to its antidepressant effect, although we observed no increases with the other antidepressants. Nevertheless, NO levels in MDD patients are reported to both increase [3], and decrease [4]. Further studies are needed to determine if MDD pathogenesis and antidepressants affect serum NO levels. Our study has several limitations. First, we investigated only the NO system in our acute depressive model. Nevertheless, changes in NOS expression and biological effects with some antidepressants were shown, even in this acute model. Comparing effective antidepressant expression changes in the NO system to investigate chronic depressive models with subacute antidepressant treatment will be performed in the future. Second, the sample size (n = 6, each group) is small, although we believe it is big enough to conclude our results, with taking animal welfare into consideration.
Conflict of interest None to declare.
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