Involvement of the neurotrophin and cannabinoid systems in the mechanisms of action of neurokinin receptor antagonists

European Neuropsychopharmacology (2011) 21, 905–917

www.elsevier.com/locate/euroneuro

Involvement of the neurotrophin and cannabinoid systems in the mechanisms of action of neurokinin receptor antagonists Parichehr Hassanzadeh a,b,⁎, Anna Hassanzadeh c a

Research Center for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran Neuropsychopharmacology Research Center, AJA University of Medical Sciences, Tehran, Iran c Department of Molecular Biology, Faculty of Molecular & Cellular Sciences, Islamic Azad University, Parand, Iran b

Received 26 July 2010; received in revised form 15 November 2010; accepted 8 January 2011

KEYWORDS Neurokinin receptor antagonists; Nerve growth factor; Cannabinoid system; Brain; Gerbil

Abstract The anxiolytic- and antidepressant-like effects of the neurokinin (NK) receptor antagonists have been shown in behavioral studies. According to the involvement of neurotrophin signaling in the mechanisms of action of psychotropic agents, we aimed to investigate whether the selective NK1, NK2, or NK3 receptor antagonists (GR-205171, SR48968, and SR142801, respectively) affect nerve growth factor (NGF) contents in the brain regions involved in the modulation of emotions. To gain a mechanistical insight into the process by which the NK antagonists regulate brain NGF levels, we evaluated the role of the cannabinoid system which is linked to depression and/or antidepressant effects and appears to interact with neurotrophin signaling. According to the results, single injection of the NK receptor antagonists (3, 5, and 10 mg/kg, i.p.) into gerbils did not alter NGF or endocannabinoid (eCB) levels quantified by Bio-Rad protein assay and isotopedilution liquid chromatography/mass spectrometry, respectively. Three-week administration of 10 mg/kg NK antagonists significantly elevated both NGF and eCB levels in brain-region specific fashion. Pre-application of the CB1 receptor neutral antagonist AM4113 (5.6 mg/kg) prevented the elevation of NGF or eCB induced by the NK antagonists. AM4113 showed no effect by itself. We conclude that the cannabinoid system is implicated in the mechanisms of action of NK receptor antagonists including the upregulation of brain NGF levels. © 2011 Elsevier B.V. and ECNP. All rights reserved.

⁎ Corresponding author at: Research Center for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Evin, P.O. Box: 19835-187, Tehran, Iran. Tel.: +98 21 22432515, +98 912 1887745 (mobile); fax: +98 21 22432517. E-mail address: [email protected] (P. Hassanzadeh).

1. Introduction In recent years, the previously dominating interest relating the effects of psychotropic medications on neurotransmitters has

0924-977X/$ - see front matter © 2011 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2011.01.002

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Figure 1 Brain regional levels of NGF at baseline and following acute administration of 3 mg/kg NK receptor antagonists. NGF levels did not differ from those of baseline values and vehicle-treated control groups (p N 0.05). A: Baseline levels of NGF, B: NGF levels 24 h after the treatment, C: NGF levels 48 h after the treatment, D: NGF levels 72 h after the treatment. NGF levels are expressed as ng of NGF per g of protein in the resuspended NGF homogenate. Data are expressed as mean ± SEM of n = 5/group.

shifted towards the effects of these agents on intraneuronal signal transduction and neurotrophins (Manji et al., 2000). There is ample evidence that neurotrophins exert numerous neuroprotective effects under pathological conditions which might be important in particular for neurodegenerative and psychiatric diseases (Shaltiel et al., 2007; Schulte-Herbrüggen et al., 2008). In general, the neurotrophic hypothesis of depression proposes that the etiology of depression and/or the action of antidepressant drugs are due, in part, to the regulation of central neurotrophin signaling. Several lines of evidence suggest that neurotrophic factors act as mediators of antidepressant responses. This has led to the investigation of the effects of psychotropic agents on neurotrophin signaling (Dias et al., 2003; Vinay et al., 2004). In recent years, the potential anxiolytic- and antidepressant-like effects of compounds that target the neurokinin (NK) receptors, a class of G proteincoupled receptors which are found in the central nervous system and peripheral tissues, have attracted a growing interest. In this sense, several selective and CNS-penetrating NK receptor antagonists which demonstrate efficacy in the treatment of emesis, anxiety, and depression have been synthesized (Dableh et al., 2005; Griebel et al., 2001; Salomé et al., 2006; Varty et al., 2002; Varty et al., 2003; Zocchi et al., 2003). As compared to the NK1 antagonists, there are limited data suggesting that the NK2 or NK3 receptor antagonists may possess antidepressant and/or anxiolytic properties, meanwhile, the existing data are promising (Ebner et al., 2009; Ribeiro et al., 1999; Rizzo et al., 2003; Steinberg et al., 2001; Stratton et al., 1993). NK1, NK2, and NK3 receptors have been identified in both rodents and humans (Bensaid et al., 2001; Pennefather et al., 2004). The localization of the NK receptors in the cortex, hippocampus, amygdala, and septum may be consistent with the anxiolyticand antidepressant-like effects of the NK antagonists. Meanwhile, the precise mechanism/s by which these therapeutic effects are brought about are not yet known. We have recently

shown that a wide range of psychotropic drugs including desipramine, fluoxetine, phenelzine, haloperidol, and clozapine elevate brain regional levels of NGF (Hassanzadeh and Hassanzadeh, 2010), however, the underlying mechanism(s) have remained elusive. In recent years, the endocannabinoid system (eCBs) and its regulatory functions in both the central and peripheral nervous systems have attracted attention. According to the reports, the eCBs is engaged in a plethora of physiological functions including the emotional disturbances (Bambico et al., 2009; Viveros et al., 2005, 2007). This ubiquitous signaling system appears to be involved in the pathophysiology and/or treatment of depression; as deficiency in the eCB signaling is associated with a behavioral phenotype similar to the symptom profile of severe depression (Hill and Gorzalka, 2005; Serra and Fratta, 2007). Furthermore, CB1 cannabinoid receptors and the enzymes involved in the synthesis and degradation of the eCB ligands are located in the brain regions crucial for emotionality and stress regulation (Vinod and Hungund, 2006; Witkin et al., 2005). Meanwhile, the neurobiological mechanism(s) linking the eCBs with the pathophysiology of mood disorders and antidepressant action remain somewhat controversial. There are several previous reports indicating the interaction between the endocannabinoids and neurotrophins as well as the signaling interaction between the CB1 and tyrosine kinase receptors (Angelucci et al., 2008; Aso et al., 2008; Calatozzolo et al., 2007; Williams et al., 2003). This background prompted us to design a study evaluating the involvement of the neurotrophin and eCB systems in the mechanisms of action of the selective NK antagonists. We selected gerbils because the structure and pharmacology of the NK receptors in gerbils resemble those of humans. In particular, gerbils have been suggested to be more suitable species than mice or rats for investigating the anxiolytic- or antidepressant-like effects of NK1 antagonists (Varty et al., 2002). Furthermore, the selective NK3 receptor antagonist osanetant has higher affinity for

Involvement of neurotrophin and CB systems in the mechanisms of action of NK receptor antagonists

Figure 2 Brain regional levels of NGF following acute administration of 5 mg/kg NK antagonists. None of the NK antagonists affected NGF levels as compared to the vehicle-treated control groups (pN 0.05). A: NGF levels 24 h after the treatment, B: NGF levels 48 h after the treatment, C: NGF levels 72 h after the treatment.

human or gerbil than for rat NK3 receptor (Emonds-Alt et al., 1995). Following treatment with the NK antagonists, we measured NGF contents in the brain regions involved in the modulation of emotions including the frontal cortex, amygdala, hippocampus, brain stem, and olfactory bulb, with a special look at the role of the cannabinoid CB1 receptors. To explore further the implication of the eCB transmission in the mechanisms of action of the NK antagonists, we measured the levels of two major endocannabinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG), in the aforementioned brain regions following administration of the NK antagonists.

2. Experimental procedures 2.1. Animals Male Mongolian gerbils weighing 220–250 g were housed in pairs under standard conditions on a 12-h light/dark cycle with ad libitum access to food pellets and water. The experiments began after at least 1 week of

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Figure 3 Brain regional levels of NGF following acute administration of 10 mg/kg NK antagonists. NGF contents remained at control levels (p N 0.05). A: NGF levels 24 h after the treatment, B: NGF levels 48 h after the treatment, C: NGF levels 72 h after the treatment. The main effects and interactions between the factors following acute administration of the NK antagonists are as follows; {frontal cortex: [dose: F(2,144) =0.21, p=0.81], [treatment: F(3,144)=1.01, p=0.39], [time point: F(2,144) = 0.69, p = 0.50], [dose × treatment: F(6,144) = 0.99, p=0.44], [dose ×time point×treatment: F(12,144)=0.24, p=0.98]; hippocampus: [dose: F(2,144)=1.56, p=0.21], [treatment: F(3,144) =1.46, p= 0.23], [time point: F(2,144)=0.03, p= 0.97], [dose ×treatment: F(6,144)=0.15, p=0.98], [dose×time point× treatment: F (12,144)=0.07, p= 1]; amygdala: [dose: F(2,144)=1.04, p=0.36], [treatment: F(3,144)= 1.52, p=0.21], [time point: F(2,144)=0.17, p=0.84], [dose ×treatment: F(6,144)=0.2; p=0.97], [dose ×time point×treatment: F(12,144)=0.09, p=1]; olfactory bulb: [dose: F (2,144)=0.05, p=0.94], [treatment: F(3,144)=0.71, P=0.54], [time point: F(2,144)=0.22, p=0.81], [dose×treatment: F(6,144)=0.33, p=0.92], [dose× time point ×treatment: F(12,144)= 0.15, p =1]; brain stem: [dose: F(2,144)= 0.32, p= 0.72], [treatment: F(3,144)= 1.31, p=0.27], [time point: F(2,144)=0.11, p=0.89], [dose ×treatment: F(6,144)=0.36, p=0.89], [dose×time point× treatment: F (12,144)=0.11, p=1]}. habituation to the housing conditions. All experimental procedures were approved by the local Ethics Committee of AJA University of Medical Sciences and carried out in accordance with the Guideline for the Care

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Table 1 Effects of the chronic administration of 3 or 5 mg/kg NK receptor antagonists on brain regional levels of NGF at different time points after the last injection. Time point (h) A 24

48

72

B 24

48

72

Brain region

Vehicle

GR205171

SR48968

SR142801

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

39.29 ± 2.61 68.52 ± 3.52 38.36 ± 2.68 40.69 ± 2.31 41.86 ± 2.89 38.70 ± 1.78 67.98 ± 3.61 38.36 ± 2.68 40.87 ± 2.18 41.77 ± 2.54 38.65 ± 2.18 68.07 ± 4.17 38.22 ± 2.45 41.78 ± 2.28 42.11 ± 2.46

44.43 ± 2.49 71.70 ± 3.64 42.14 ± 3.11 42.65 ± 2.89 48.87 ± 2.86 42.61 ± 3.42 74.67 ± 3.03 45.57 ± 2.59 46.43 ± 2.73 43.52 ± 2.46 43.66 ± 2.11 68.53 ± 2.96 46.11 ± 3.10 47.26 ± 2.33 44.06 ± 3.18

50.91 ± 2.93 77.52 ± 3.69 47.56 ± 2.67 46.81 ± 2.59 43.26 ± 3.09 49.28 ± 3.53 70.12 ± 2.74 42.78 ± 2.99 49.11 ± 3.09 46.96 ± 2.18 46.38 ± 3.04 74.04 ± 3.74 49.57 ± 3.21 49.53 ± 3.26 42.52 ± 2.96

43.62 ± 2.82 70.79 ± 3.85 46.79 ± 2.26 49.12 ± 2.37 47.55 ± 2.36 47.04 ± 2.87 76.72 ± 3.72 49.24 ± 2.58 43.64 ± 2.64 48.29 ± 3.37 47.41 ± 2.28 76..35 ± 4.02 47.29 ± 2.35 43.68 ± 3.33 46.94 ± 3.58

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

40.69 ± 2.51 68.52 ± 3.52 39.46 ± 2.48 42.26 ± 2.82 42.99 ± 2.54 40.96 ± 2.11 68.12 ± 3.04 39.46 ± 2.48 40.68 ± 2.08 42.47 ± 2.46 40.95 ± 2.43 66.71 ± 2.88 39.23 ± 2.72 40.23 ± 2.21 43.47 ± 2.87

45.86 ± 2.82 72.98 ± 2.94 42.29 ± 2.54 46.27 ± 2.86 47.38 ± 2.71 46.15 ± 3.06 72.30 ± 2.36 43.35 ± 2.64 45.64 ± 2.87 47.10 ± 2.933 44.82 ± 2.57 70.22 ± 3.26 44.23 ± 2.57 44.79 ± 2.74 46.94 ± 2.68

43.41 ± 3.15 76.77 ± 3.85 47.13 ± 3.09 42.68 ± 2.69 44.02 ± 3.12 45.66 ± 3.01 74.22 ± 3.51 44.75 ± 2.35 46.83 ± 2.16 44.79 ± 3.09 47.78 ± 2.38 71.64 ± 2.87 42.64 ± 2.65 45.62 ± 2.36 46.80 ± 2.53

50.25 ± 3.04 73.51 ± 3.86 50.08 ± 2.65 43.90 ± 3.05 42.35 ± 2.18 47.39 ± 2.73 69.54 ± 2.99 47.28 ± 2.97 44.31 ± 2.38 43.84 ± 2.76 46.18 ± 2.69 68.20 ± 3.12 46.81 ± 3.08 41.48 ± 2.34 45.32 ± 3.03

A: Treatment with 3 mg/kg NK antagonists, B: Treatment with 5 mg/kg NK antagonists. NGF levels are expressed as ng of NGF per g of protein in the resuspended NGF homogenate. Data expressed as mean ± SEM of n = 5/group.

and Use of Laboratory Animals as adopted by the National Institutes of Health (NIH Publications No. 8023, revised 1978).

2.2. Drug treatments The selective NK1, NK2, and NK3 receptor antagonists: GR205171 ([2methoxy-5-(5-trifluoromethyl-tetrazol-1-yl)-benzyl]-(2S-phenylpiperidin-3S-yl)-amine), (Glaxo Smith Kline-Beecham, UK); SR48968 (saredutant), {(S)-N-methyl-N-[4-(4-acetylamino-4-phenylpiperidino)2-(3,4-dichloro phenyl)butyl]Benzamide}; and SR142801 (osanetant), N-[1-[3[1-benzoyl-3-(3,4-dichlorophenyl)-3-piperidinyl]propyl]-4phenyl-4-piperidinyl]-N-methyl-monohydrochloride (Sanofi-Synthelabo Recherche, France) were dissolved in physiological saline containing 0.1% Tween 80 (Sigma-Aldrich, Germany) and administered intraperitoneally (i.p.) at doses of 3, 5, and 10 mg/kg (Dableh et al., 2005; Griebel et al., 2001; Salomé et al., 2006; Varty et al., 2003; Zocchi et al., 2003) in a volume of 1 ml/kg. Animals received drug or vehicle once daily between 9:00 and 10:00 a.m. for either 1 day or 21 consecutive days (n=5/group). Route of administration and duration of treatment were selected based on the previous studies showing the

regulation of NGF mRNA, and brain-derived neurotrophic factor (BDNF) or fibroblast growth factor (FGF) protein by psychotropic medications (Angelucci et al., 2000; Alvin et al., 2007; Dias et al., 2003; Hassanzadeh and Hassanzadeh, 2010; Vinay et al., 2004). In case of NGF alteration due to the acute or chronic administration of NK antagonists, the CB1 receptor neutral antagonist AM4113 (N-piperidin-1-yl-2,4-dichlorophenyl-1H-pyrazole-3-carboxamide analog, Center for Drug Discovery, Northeastern University, USA) was dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich, Germany), Tween-80, and 0.9% saline in a 1:1:8 ratio and injected daily at doses of 1, 3, and 5.6 mg/kg, i.p. (Chambers et al., 2007; Järbe et al., 2008; Sink et al., 2008) 30 min prior to the injection of NK antagonist (n =5/group).

2.3. NGF quantification Brain regional levels of NGF were measured at three time points (24, 48 and 72 h) after the last injection of drug or vehicle (Alvin et al., 2007; Angelucci et al., 2000; Dias et al., 2003; Gwinn et al., 2002; Vinay et al., 2004). Animals were decapitated without anesthesia and the brain of each animal was quickly and carefully removed from

Involvement of neurotrophin and CB systems in the mechanisms of action of NK receptor antagonists

Figure 4 Effects of the chronic administration of NK antagonists (10 mg/kg) on brain regional levels of NGF. Three-week administration of the NK antagonists led to the elevation of NGF levels in a brain region-specific fashion. A: NGF levels 24 h after the treatment, B: NGF levels 48 h after the treatment, C: NGF levels 72 h after the treatment. None of the drugs significantly altered NGF level in the brain stem. The main effects and interactions between the factors are as follows; [dose: F(2,144)= 1.27, p = 0.29], [treatment: F(3,144) = 1.25, p = 0.29], [time point: F(2,144) = 0.07, p = 0.93], [dose × treatment: F(6,144) = 0.1, p = 0.99], [dose × time point × treatment: F(12,144)= 0.19, p= 0.99]. Data are expressed as mean ± SEM of n = 5/group. ⁎p b 0.05, ⁎⁎p b 0.01, and ⁎⁎⁎p b 0.001 as compared to the vehicle-treated control groups.

the skull. The frontal cortex, hippocampus, amygdala, olfactory bulb, and brain stem were dissected on a frozen pad taken from a −80 °C freezer. All tissues were immediately frozen at − 80 °C. In homogenization procedure, tissue samples were individually homogenized on ice in 5 to 6 volumes of 0.25 mol/L sucrose, and 10 mmol/L HEPES (pH 7.0) containing 10 mmol/L DTT, then, immediately were frozen in a dry ice/acetone bath and stored at −80 °C until NGF analysis. As previously reported, NGF content in adult rat brain is several fold higher than generally reported and is largely associated with sediment-

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Figure 5 Effect of AM4113 on the enhancement of NGF induced by the NK antagonists. Daily pretreatment with AM4113 at doses of 1 mg/kg (A) or 3 mg/kg (B) did not prevent the elevation of NGF levels, while, 5.6 mg/kg AM4113 (C) showed a preventive effect in this regard (p N 0.05). Vehicles 1 and 2 are related to the NK antagonists and AM4113, respectively. (GR2: GR205171, SR4: SR48968, SR1: SR142801, AM/…: pretreatment with AM4113). Data are expressed as mean ± SEM of n = 5/group. ⁎p b 0.05.

able fractions (Hoener et al., 1996), therefore, the homogenates were centrifuged at 10,000×g for 10 min at 15 °C and the remaining pellets were dissolved in 750 μl NGF homogenization buffer, treated with ultrasound for 3 min, and processed for quantification of NGF as previously described in detail (Hellweg et al., 1989, 2002). The measured and recovery-corrected NGF contents are expressed in ng NGF per g protein in the resuspended NGF homogenate quantified by Bradford protein assay (Bio-Rad, Hercules, USA) (Bradford, 1976). All the measurements were performed in duplicate and analyzed by an investigator blind to the experimental set-up.

2.4. Endocannabinoid extraction and analysis In order to explore the implication of eCB transmission in the action of NK receptor antagonists, we measured the levels of two major endocannabinoids, AEA and 2-AG, in the aforementioned brain regions following treatment with NK antagonists. Animals were

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Figure 6 Effect of AM4113 on brain regional levels of NGF. Acute or 21-day treatment with AM4113 (5.6 mg/kg) alone did not alter NGF content in any brain region analyzed (p N 0.05). A: Single injection of AM4113, B: Chronic treatment with AM4113. Data represent means ± SEM of n = 5/group.

sacrificed 1, 5, and 12 h after the last injection of drug or vehicle (De Lago et al., 2005) and the same brain regions were dissected as mentioned above (n = 5/group). Brain tissue samples were subjected to a lipid extraction process (Patel et al., 2003) and the contents of AEA and 2-AG within the lipid extracts were determined using isotope-dilution liquid chromatography/mass spectrometry (Koga et al., 1997). The amounts of endocannabnoids are expressed as pmol or nmol per gram of wet tissue extracted. The measurements were performed in duplicate and analyzed by an investigator blind to the experimental set-up.

2.5. Statistical analysis The Kolmogorov–Smirnov test was used to verify normal distribution of the experimental data. Brain regional levels of NGF or endocannabinoids were analyzed by three-way ANOVA followed by the Tukey's post hoc analysis (StatView 5 software; SAS Institute Inc., Cary, NC). Data are presented as mean ± SEM (5 animals per group). The level of significance was set at p b 0.05.

3. Results 3.1. Effects of the NK receptor antagonists on brain regional levels of NGF Acute administration of NK1, NK2, or NK3 receptor antagonists (3 mg/kg) did not alter NGF levels at any time point tested as compared with baseline values or vehicle-treated control groups (Fig. 1, p N 0.05). Acute treatment with the NK antagonists at doses of 5 or 10 mg/kg did not produce any significant change (Figs. 2 and 3, p N 0.05. The main effects and interactions between the factors are provided in the figure legends).

P. Hassanzadeh, A. Hassanzadeh Chronic administration of the NK antagonists at doses of 3 or 5 mg/kg did not affect brain regional levels of NGF at any time point tested (Table 1A and B, p N 0.05), while, 24 h after the last injection of all three NK antagonists at a daily dose of 10 mg/kg, post hoc comparisons revealed a significant elevation of NGF contents in the frontal cortex and hippocampus (Fig. 4A) which remained significantly elevated as long as 48 and 72 h (Fig. 4B and C, respectively). The main effects and interactions between the factors are as follows; frontal cortex: [dose: F (2,144) = 17.45, p b 0.05], [treatment: F(3,144) = 17.81, p b 0.05], [time point: F(2,144) = 0.37, p = 0.69], [dose × treatment: F(6,144) = 3.01, p = 0.02], [dose × time point × treatment: F (12,144) = 0.47, p = 0.93]; hippocampus: [dose: F(2,144) = 38.47, p b 0.05], [treatment: F(3,144) = 12.58, p b 0.05], [time point: F (2,144) = 2.18, p = 0.12], [dose × treatment: F(6,144) = 3.45, p = 0.003], [dose × time point × treatment: F(12,144) = 0.42, p = 0.96]}. According to the Tukey's post hoc test, both SR48968 and SR142801 significantly elevated NGF concentration in the amygdala and olfactory bulb at all time points tested (Fig. 4A, B, and C), whereas, GR205171 showed no effect (Fig. 4, pN 0.05). The main effects and interactions between the factors are as follows; amygdala: [dose: F(2,144)= 8.31, p b 0.05], [treatment: F(3,144)= 35.31, pb 0.05], [time point: F(2,144)=0.14, p=0.87], [dose× treatment: F(6,144)=5.52, pb 0.05], [dose× time point ×treatment: F (12,144)=0.44, p=0.95]; olfactory bulb: [dose: F(2,144)=9.65, pb 0.05], [treatment: F(3,144)=16.73, p b 0.05], [time point: F (2,144) = 0.01, p = 0.99], [dose × treatment: F(6,144) = 3.81, p = 0.001], [dose × time point × treatment: F(12,144) = 0.26, p= 0.99]. None of the drugs significantly altered NGF level in the brain stem (Fig. 4, pN 0.05).

3.2. The CB1 receptor neutral antagonist dosedependently blocks NK antagonists-mediated enhancement of NGF Daily pretreatment with 1 or 3 mg/kg AM4113 did not affect the enhanced levels of NGF induced by the NK antagonists (Fig. 5A and B, p b 0.05), while, 5.6 mg/kg AM4113 showed a preventive effect in this regard (Fig. 5C, p N 0.05).

3.3. AM4113 alone does not affect brain regional levels of NGF Acute or 21-day treatment with AM4113 (5.6 mg/kg) did not alter NGF content in any brain region analyzed (Fig. 6A and B, p N 0.05).

3.4. Effects of the NK antagonists on brain regional contents of endocannabinoids 1 h after the acute administration of 3 mg/kg NK antagonists, brain regional levels of endocannabinoids did not differ significantly from those at baseline or vehicle groups (Table 2A and B, p N 0.05). Similar results were obtained following acute administration of the NK antagonists at doses of 5 or 10 mg/kg (Table 3A and B, p N 0.05). Endocannabinoid contents remained unchanged up to 5 and 12 h (not shown). 1 h after the chronic administration of 3 or 5 mg/kg NK antagonists, the eCB contents did not change significantly in any brain region examined (Table 4A and B, p N 0.05). Similar results were obtained at 5 and 12 h (not shown). Chronic administration of 10 mg/kg NK antagonists led to

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Table 2 Brain regional levels of endocannabinoids at baseline and following acute administration of 3 mg/kg NK receptor antagonists. A Brain region

AEA (pmol/g tissue)

2-AG (nmol/g tissue)

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

6.86 ± 0.56 18.10 ± 0.99 5.87 ± 0.51 6.35 ± 0.67 5.74 ± 0.52

2.95 ± 0.36 5.21 ± 0.48 4.99 ± 0.61 4.17 ± 0.44 2.05 ± 0.29

B Endocannabinoid

Brain region

Vehicle

GR205171

SR48968

SR142801

AEA (pmol/g tissue)

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.15 ± 0.67 18.51 ± 0.82 5.92 ± 0.44 6.31 ± 0.69 6.21 ± 0.57 3.48 ± 0.39 5.22 ± 0.54 4.96 ± 0.47 4.55 ± 0.49 2.15 ± 0.28

7.22 ± 0.61 18.76 ± 0.94 6.19 ± 0.42 6.21 ± 0.58 6.32 ± 0.61 3.93 ± 0.35 5.15 ± 0.49 4.81 ± 0.46 4.86 ± 0.51 2.69 ± 0.21

6.56 ± 0.64 19.11 ± 1.14 6.19 ± 0.43 6.58 ± 0.53 7.17 ± 0.76 3.26 ± 0.37 5.72 ± 0.49 5.09 ± 0.59 5.03 ± 0.56 3.83 ± 0.32

6.92 ± 0.56 18.48 ± 0.96 5.87 ± 0.51 6.36 ± 0.52 6.13 ± 0.52 3.81 ± 0.34 5.17 ± 0.51 5.14 ± 0.59 4.64 ± 0.49 2.37 ± 0.31

2-AG (nmol/g tissue)

A: Baseline levels of endocannabinoids, B: Endocannabinoid contents 1 h after administration of the NK antagonists. Data are expressed as mean ± SEM of n = 5/group.

a region-specific elevation of both AEA and 2-AG contents in gerbil brain at 1 h from the last injection that lasted for up to 5 and 12 h (Table 5A, B, and C, p b 0.05 and p b 0.01 versus vehicle-treated groups. The main effects and interaction between the factors are provided in the table legend). Table 3

3.5. AM4113 dose-dependently blocks the elevation of endocannabinoids induced the NK antagonists Daily pretreatment with 1 or 3 mg/kg AM4113 did not affect the elevated levels of endocannabinoids induced by the chronic

Brain regional levels of endocannabinoids following acute administration of 5 or 10 mg/kg NK receptor antagonists.

Endocannabinoid A AEA (pmol/g tissue)

2-AG (nmol/g tissue)

B AEA (pmol/g tissue)

2-AG (nmol/g tissue)

Brain region

Vehicle

GR205171

SR48968

SR142801

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.15 ± 0.67 18.51 ± 0.82 5.92 ± 0.44 6.31 ± 0.69 6.21 ± 0.57 3.48 ± 0.39 5.22 ± 0.54 4.96 ± 0.47 4.55 ± 0.49 2.15 ± 0.28

6.48 ± 0.71 20.17 ± 1.96 5.63 ± 0.51 7.12 ± 0.76 5.86 ± 0.62 3.24 ± 0.38 6.13 ± 0.57 4.61 ± 0.53 5.13 ± 0.57 2.07 ± 0.27

7.37 ± 0.68 19.24 ± 1.32 7.05 ± 0.73 5.83 ± 0.49 6.48 ± 0.64 4.11 ± 0.47 5.17 ± 0.61 5.09 ± 0.59 5.03 ± 0.56 2.46 ± 0.31

6.72 ± 0.63 17.94 ± 1.46 6.23 ± 0.54 5.94 ± 0.61 6.47 ± 0.59 3.81 ± 0.34 6.07 ± 0.63 5.14 ± 0.54 4.64 ± 0.49 2.37 ± 0.24

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.15 ± 0.67 18.51 ± 0.82 5.92 ± 0.44 6.31 ± 0.69 6.21 ± 0.57 3.48 ± 0.39 5.22 ± 0.54 4.96 ± 0.47 4.55 ± 0.49 2.15 ± 0.28

7.43 ± 0.76 18.06 ± 1.37 5.47 ± 0.53 6.49 ± 0.58 7.06 ± 0.77 3.62 ± 0.33 5.37 ± 0.44 5.13 ± 0.56 4.32 ± 0.43 2.37 ± 0.23

6.56 ± 0.64 19.46 ± 1.76 6.32 ± 0.64 6.14 ± 0.64 5.73 ± 0.62 3.27 ± 0.37 6.08 ± 0.65 5.47 ± 0.63 5.17 ± 0.41 2.29 ± 0.27

6.92 ± 0.56 18.25 ± 1.29 5.35 ± 0.47 6.72 ± 0.67 5.48 ± 0.64 4.12 ± 0.52 5.35 ± 0.48 4.47 ± 0.42 5.28 ± 0.57 2.08 ± 0.23

Data were obtained 1 h after the injection of NK antagonists or vehicle. A: Treatment with 5 mg/kg NK antagonists, B: Treatment with 10 mg/kg NK antagonists.

912 Table 4

P. Hassanzadeh, A. Hassanzadeh Endocannabinoid contents in brain regions of gerbils chronically exposed to 3 or 5 mg/kg NK receptor antagonists.

Endocannabinoid A AEA (pmol/g tissue)

2-AG (nmol/g tissue)

B AEA (pmol/g tissue)

2-AG (nmol/g tissue)

Brain region

Vehicle

GR205171

SR48968

SR142801

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.38 ± 0.61 19.18 ± 1.06 6.19 ± 0.44 6.41 ± 0.69 5.93 ± 0.69 3.23 ± 0.29 5.14 ± 0.51 5.41 ± 0.54 4.29 ± 0.42 2.29 ± 0.23

8.55 ± 0.54 21.25 ± 0.88 6.49 ± 0.37 6.72 ± 0.47 6.22 ± 0.56 3.71 ± 0.37 5.39 ± 0.37 5.37 ± 0.48 4.59 ± 0.37 2.12 ± 0.31

8.59 ± 0.59 20.91 ± 0.95 6.36 ± 0.39 7.07 ± 0.65 7.16 ± 0.82 3.66 ± 0.36 4.97 ± 0.51 5.47 ± 0.54 5.23 ± 0.56 3.04 ± 0.31

8.56 ± 0.61 21.67 ± 1.12 6.53 ± 0.79 7.18 ± 0.87 6.13 ± 0.52 3.54 ± 0.34 5.57 ± 0.42 6.06 ± 0.66 4.69 ± 0.42 3.11 ± 0.43

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.31 ± 0.56 19.59 ± 1.07 6.05 ± 0.52 6.65 ± 0.68 5.66 ± 0.69 3.02 ± 0.34 5.35 ± 0.59 4.95 ± 0.51 4.38 ± 0.46 2.18 ± 0.25

9.11 ± 0.69 21.41 ± 0.99 6.53 ± 0.63 7.07 ± 0.68 6.18 ± 0.51 3.89 ± 0.37 5.65 ± 0.51 5.59 ± 0.54 4.90 ± 0.38 3.02 ± 0.32

9.15 ± 0.82 22.08 ± 1.20 7.09 ± 0.74 7.16 ± 0.72 7.34 ± 0.57 3.79 ± 0.36 5.54 ± 0.66 5.94 ± 0.46 5.07 ± 0.49 3.35 ± 0.31

9.09 ± 0.62 21.31 ± 1.10 7.11 ± 0.65 7.62 ± 0.61 6.49 ± 0.69 3.59 ± 0.39 6.01 ± 0.61 5.65 ± 0.63 5.18 ± 0.49 3.27 ± 0.42

Data were obtained 1 h after the last injection in 3-week daily drug administration. A: Treatment with 3 mg/kg NK antagonists, B: Treatment with 5 mg/kg NK antagonists. Data are expressed as mean ± SEM of n = 5/group.

treatment with 10 mg/kg NK antagonists (Table 6A and B, p b 0.05), while, 5.6 mg/kg AM4113 showed a preventive effect in this regard (Table 6C, p N 0.05).

4. Discussion Over the past decade, increased neurogenesis, neuroplasticity, and neuroprotection have become the focus of intense research especially in neuropsychiatric disorders. In this sense, contribution of the endogenous neurotrophins in the action of antidepressant drugs has been considered as an exciting new mechanism of action of these drugs (Berton and Nestler, 2006; Hassanzadeh and Hassanzadeh, 2010). In recent years, the anxiolytic- and antidepressant-like effects of NK receptor antagonists have been shown in several types of behavioral testing including the forced swim test, elevated plus-maze, and social interaction tests (Dableh et al., 2005; Gentsch et al., 2002; Varty et al., 2002; Zocchi et al., 2003). There are limited data suggesting that NK2 or NK3 receptor antagonists may possess antidepressant and/or anxiolytic properties as compared to the NK1 antagonists. Meanwhile, according to Ribeiro et al. (1999), NK3 receptors may be involved in anxiety. In other reports, blockade of the NK2 receptors have been shown to induce antidepressantand/or anxiolytic activities (Steinberg et al., 2001; Stratton et al., 1993). In parallel, efforts have been made for the development of dual and triple NK receptor antagonists (Rizzo et al., 2003). However, the effects of NK antagonists on neurotrophic factors are poorly characterized as com-

pared to the commonly used psychotropic medications. There are a few studies that have shown the differential regulation of NK1 receptor and BDNF gene expression during inflammatory pain (Duric and McCarson, 2007) or the increased levels of BDNF in NK1 receptor gene knockout mice (Morcuende et al., 2003), however, there is no report linking the NK antagonists to NGF. According to our findings, acute treatment with the NK antagonists at all doses tested did not significantly alter NGF levels (Figs. 1–3). Meanwhile, it has been previously shown that acute administration of the NK1 receptor antagonist, NKP608, provoke anxiolytic-like effect (Vendruscolo et al., 2003). In general, at least two separate processes should be considered in order to understand the mechanism(s) of antidepressant action; 1) an early process, and 2) a slowly developing process (Castrén, 2004; Katz et al., 1997). The latter process appears to include the neurotrophic effects of antidepressant treatment, since the increased expression of a trophic factor is usually considered as a slow-onset adaptive change (Yanpallewar et al., 2010). In this context, the upregulation of neurotrophins has been shown to occur in response to chronic, but not acute, antidepressant treatment (Adachi et al., 2008; Dranovsky and Hen, 2006; Malberg et al., 2000; Shirayama et al., 2002). As shown in Fig. 4, chronic treatment with the selective NK1, NK2, or NK3 antagonists led to the elevation of NGF in a dose-dependent and brain region-specific fashion. Repeated injections of all three NK antagonists significantly increased NGF level in the frontal cortex (Fig. 4). In an anatomical point of view, the frontal cortex is likely to be

Involvement of neurotrophin and CB systems in the mechanisms of action of NK receptor antagonists Table 5

913

Endocannabinoid contents in brain regions of gerbils chronically exposed to 10 mg/kg NK receptor antagonists.

Endocannabinoid A AEA (pmol/g tissue)

2-AG (nmol/g tissue)

B AEA (pmol/g tissue)

2-AG (nmol/g tissue)

C AEA (pmol/g tissue)

2-AG (nmol/g tissue)

Brain region

Vehicle

GR205171

SR48968

SR142801

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.31 ± 0.59 19.34 ± 1.03 6.15 ± 0.41 6.21 ± 0.54 5.84 ± 0.45 3.11 ± 0.32 5.19 ± 0.51 5.18 ± 0.49 4.42 ± 0.48 2.34 ± 0.22

10.97 ± 0.84 ⁎ 25.81 ± 1.39 ⁎ 6.23 ± 0.68 7.03 ± 0.52 6.26 ± 0.41 5.33 ± 0.37 ⁎ 8.42 ± 0.53 ⁎⁎

11.63 ± 0.98 ⁎⁎ 25.46 ± 1.54 ⁎ 9.57 ± 0.61 ⁎⁎ 7.01 ± 0.69 7.61 ± 0.55 5.33 ± 0.55 ⁎ 7.77 ± 0.45 ⁎

5.41 ± 0.44 5.21 ± 0.45 3.04 ± 0.29

5.73 ± 0.51 7.19 ± 0.88 ⁎ 3.48 ± 0.28

12.46 ± 0.89 ⁎⁎ 25.96 ± 1.38 ⁎ 7.13 ± 0.85 9.01 ± 0.77 ⁎ 6.64 ± 0.62 5.16 ± 0.58 ⁎ 8.16 ± 0.84 ⁎ 7.76 ± 0.70 ⁎ 7.48 ± 0.85 ⁎

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.31 ± 0.59 19.34 ± 1.03 6.15 ± 0.41 6.21 ± 0.54 5.84 ± 0.45 3.11 ± 0.32 5.19 ± 0.51 5.18 ± 0.49 4.42 ± 0.48 2.34 ± 0.22

10.45 ± 0.79 ⁎ 26.53 ± 1.47 ⁎ 5.97 ± 0.56 6.49 ± 0.58 5.32 ± 0.47 5.27 ± 0.31 ⁎ 7.58 ± 0.57 ⁎

10.93 ± 0.82 ⁎ 24.97 ± 1.26 ⁎ 9.98 ± 0.72 ⁎ 6.92 ± 0.63 6.35 ± 0.61 6.78 ± 0.51 ⁎⁎ 8.73 ± 0.85 ⁎

5.07 ± 0.47 4.73 ± 0.41 2.86 ± 0.26

5.16 ± 0.44 6.84 ± 0.66 ⁎ 3.16 ± 0.37

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.31 ± 0.59 19.34 ± 1.03 6.15 ± 0.41 6.21 ± 0.54 5.84 ± 0.45 3.11 ± 0.32 5.19 ± 0.51 5.18 ± 0.49 4.42 ± 0.48 2.34 ± 0.22

11.68 ± 0.82 ⁎⁎ 24.73 ± 1.22 ⁎ 6.39 ± 0.54 6.14 ± 0.57 5.41 ± 0.48 6.49 ± 0.58 ⁎ 7.85 ± 0.63 ⁎ 5.48 ± 0.53 5.34 ± 0.56 3.17 ± 0.31

11.21 ± 0.72 ⁎⁎ 24.39 ± 1.18 ⁎ 8.13 ± 0.76 ⁎ 5.79 ± 0.61 6.28 ± 0.53 5.51 ± 0.56 ⁎ 9.78 ± 0.84 ⁎ 5.83 ± 0.58 8.96 ± 0.77 ⁎

10.74 ± 0.73 ⁎ 26.35 ± 1.42 ⁎ 6.83 ± 0.62 9.36 ± 0.79 ⁎

2.94 ± 0.26

3.14 ± 0.38

3.41 ± 0.35

11.59 ± 0.76 ⁎ 26.15 ± 1.42 ⁎ 6.58 ± 0.57 9.86 ± 0.65 ⁎⁎ 5.93 ± 0.58 5.79 ± 056 ⁎ 8.29 ± 0.79 ⁎ 7.83 ± 0.61 ⁎ 8.76 ± 0.64 ⁎⁎ 2.93 ± 0.26

5.36 ± 0.49 6.34 ± 0.62 ⁎ 7.69 ± 0.68 ⁎ 7.66 ± 0.62 ⁎ 6.83 ± 0.58 ⁎

Data were obtained 1 h (A), 5 h (B), and 12 h (C) after the last injection in 3-week daily drug administration. The main effects and interaction between the factors are as follows; {frontal cortex (2-AG): [dose: F(2,144)=32.51, pb 0.05], [treatment: F(3,144)=13.68, pb 0.05], [time point: F(2,144)=0.08, p =0.93], [dose×treatment: F(6,144)=3.78, p=0.002], [dose×time point×treatment: F(12,144)=0.04, p =1]; frontal cortex (AEA): [dose: F(2,144)=27.51, pb 0.05], [treatment: F(3,144)=24.15, pb 0.05], [time point: F(2,144)=0.44, p =0.65], [dose×treatment: F(6,144)=3.69, p =0.002], [dose×time point×treatment: F(12,144)=0.98, p=1]; hippocampus (2-AG): [dose: F(2,144)=58.61, pb 0.05], [treatment: F(3,144)= 9.42, pb 0.05], [time point: F(2,144)=0.41, p =0.67], [dose×treatment: F(6,144)=6.31, pb 0.05], [dose×time point ×treatment: F(12,144)= 0.05, p=1]; hippocampus (AEA): [dose: F(2,144)=32.14, pb 0.05], [treatment: F(3,144)=9.97, p b 0.05], [time point: F(2,144)=0.82, p=0.44], [dose×treatment: F(6,144)=3.78, p =0.002], [dose×time point×treatment: F(12,144)=0.22, p=0.99]; amygdala (2-AG): [dose: F(2,144)=6.23, p =0.003], [treatment: F(3,144)=5, p=0.003], [time point: F(2,144)=0.32, p =0.73], [dose×treatment: F(6,144)=5.24, pb 0.05], [dose×time point×treatment: F(12,144)=0.05, p =1]; amygdala (AEA): [dose: F(2,144)=9.481, p b 0.05], [treatment: F(3,144)=19.22, p b 0.05], [time point: F(2,144)=0.44, p=0.64], [dose×treatment: F(6,144)=6.7, pb 0.05], [dose×time point×treatment: F(12,144)=0.88, p=1]; olfactory bulb (2-AG): [dose: F(2,144)=18.57, pb 0.05], treatment: F(3,144)=9.37, pb 0.05], [time point: F(2,144)=0.16, p=0.85], [dose×treatment: F(6,144)=4.76, p b 0.05], [dose×time point×treatment: F(12,144)=0.14, p=1]; olfactory bulb (AEA): [dose: F(2,144)=3.85, p=0.02], [treatment: F(3,144)=4.89, p =0.003], [time point: F(2,144)=0.18, p=0.84], [dose×treatment: F(6,144)=2.58, p=0.02], [dose×time point ×treatment: F(12,144)=0.25, p =0.99]; brain stem (2-AG): [dose: F(2,144)=1.85, p =0.16], [treatment: F(3,144)=1.46, p=0.23], [time point: F(2,144)=0.75, p =0.48], [dose×treatment: F(6,144)=0.64, p =0.69], [dose×time point×treatment: F(12,144)=0.46, p=0.94]; brain stem (AEA): [dose: F(2,144)=0.34, p =0.71], [treatment: F(3,144)=2.35, p=0.08], [time point: F(2,144)=0.003, p =0.99], [dose×treatment: F(6,144)=0.32, p=0.93], [dose×time point×treatment: F(12,144)=0.2, p=0.99]}. Data are expressed as mean ± SEM of n = 5/group. ⁎ p b 0.05. ⁎⁎ p b 0.01.

914

P. Hassanzadeh, A. Hassanzadeh

Table 6 Effect of AM4113 on the endocannabinoid contents in brain regions of gerbils chronically exposed to the NK receptor antagonists. Endocannabinoid A AEA (pmol/g tissue)

2-AG (nmol/g tissue)

B AEA (pmol/g tissue)

2-AG (nmol/g tissue)

C AEA (pmol/g tissue)

2-AG (nmol/g tissue)

Brain region

Vehicle

AM/GR205171

AM/SR48968

AM/SR142801

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.31 ± 0.59 19.34 ± 1.03 6.15 ± 0.41 6.21 ± 0.54 5.84 ± 0.45 3.11 ± 0.32 5.19 ± 0.51 5.18 ± 0.49 4.42 ± 0.48 2.34 ± 0.22

11.21 ± 0.89⁎ 26.52 ± 1.46⁎ 6.44 ± 0.59 5.84 ± 0.48 5.69 ± 0.53 6.17 ± 0.48* 7.58 ± 0.53* 5.07 ± 0.47 4.73 ± 0.41 3.16 ± 0.34

10.47 ± 0.76⁎ 25.19 ± 1.37⁎ 10.08 ± 0.72⁎ 6.14 ± 0.57 5.37 ± 0.49 5.56 ± 0.51* 8.67 ± 0.64* 5.16 ± 0.44 6.84 ± 0.63* 2.45 ± 0.26

10.38 ± 0.78⁎ 26.31 ± 1.43⁎ 6.58 ± 0.57 9.45 ± 0.74* 6.31 ± 0.57 5.48 ± 0.56* 7.87 ± 0.63* 7.83 ± 0.67* 6.94 ± 0.59* 3.29 ± 0.37

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.31 ± 0.59 19.34 ± 1.03 6.15 ± 0.41 6.21 ± 0.54 5.84 ± 0.45 3.11 ± 0.32 5.19 ± 0.51 5.18 ± 0.49 4.42 ± 0.48 2.34 ± 0.22

10.27 ± 0.72⁎ 25.49 ± 1.37⁎ 5.43 ± 0.53 6.09 ± 0.56 6.17 ± 0.51 5.83 ± 0.42⁎ 7.62 ± 0.53⁎ 5.09 ± 0.46 4.66 ± 0.41 3.07 ± 0.27

10.07 ± 068⁎ 25.18 ± 1.12⁎ 9.45 ± 0.69⁎ 5.64 ± 0.49 5.46 ± 0.47 6.14 ± 0.55⁎ 8.13 ± 0.67⁎ 5.28 ± 0.53 6.72 ± 0.57⁎

10.76 ± 0.69⁎ 24.83 ± 1.28⁎ 6.73 ± 0.59 9.24 ± 0.73⁎

3.11 ± 0.31

2.82 ± 0.29

Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem Frontal cortex Hippocampus Amygdala Olfactory bulb Brain stem

7.31 ± 0.59 19.34 ± 1.03 6.15 ± 0.41 6.21 ± 0.54 5.84 ± 0.45 3.11 ± 0.32 5.19 ± 0.51 5.18 ± 0.49 4.42 ±0.48 2.34 ± 0.22

8.12 ± 0.59 19.89 ± 1.04 6.58 ± 0.52 6.04 ± 0.48 6.04 ± 0.41 4.61 ± 0.59 6.11 ± 0.55 5.01 ± 0.54 4.81 ± 0.41 2.43 ± 0.21

7.71 ± 0.75 20.52 ± 1.08 6.84 ± 0.59 6.93 ± 0.68 7.06 ± 0.68 4.87 ± 0.43 6.21 ± 0.66 5.73 ± 0.51 3.87 ± 0.43 2.98 ± 0.27

8.21 ± 0.57 21.14 ± 1.52 7.59 ± 0.58 7.79 ± 0.69 6.58 ± 0.54 3.78 ± 0.39 5.54 ± 0.55 4.91 ± 0.49 4.93 ± 0.45 3.07 ± 0.34

5.73 ± 0.55 5.18 ± 0.53⁎ 8.27 ± 0.78⁎ 7.93 ± 0.72⁎ 8.11 ± 0.77⁎

Daily pretreatment with the CB1 receptor neutral antagonist AM4113 at doses of 1 mg/kg (A) or 3 mg/kg (B) did not prevent the elevation of endocannabinoids induced by the NK antagonists (p ≤ 0.05), while, the eCB contents remained at control levels due to the pre-application of 5.6 mg/kg AM4113 (C, p ≥ 0.05). Data were obtained at 1 h from the last injection of NK receptor antagonists and expressed as mean ± SEM of n = 5/group. (AM/…: pretreatment with AM4113). * p b 0.05.

involved in depression and antidepressant medications have been suggested to block or reverse stress-induced pathogenic deficits in this brain area (Banasr et al., 2007; Duman, 2004). According to the stimulatory effect of NGF on cell proliferation (Cheng et al., 2009), enhancement of the frontal cortex NGF by NK antagonists may be of therapeutic importance in stress-induced reduction in cell proliferation. The NK antagonists also elevated NGF concentration in the hippocampus (Fig. 4), which is supposed to be indicative of their neuroprotective effects. As known, NGF plays a critical role in the hippocampal plasticity and learning and regulates hippocampal neurogenesis, a plastic process that is regulated in response to the antidepressant treatment (Conner et al., 2009). Furthermore, NGF is involved in cognitive function via the induction of acetylcholine release in the

hippocampus (Winkler et al., 2000). It is therefore reasonable to speculate that NK antagonists improve psychopathology and particularly cognitive performance via the enhancement of hippocampal NGF. As previously suggested, NK antagonists may exert neuroprotective effects through different mechanisms (Vink et al., 2001). In a recently conducted study, the NK1 antagonist, N-acetyl-L-tryptophan, improved the cognitive neurologic outcomes after traumatic brain injury (Donkin et al., 2009). In other CNS areas for NGF production; olfactory bulb and amygdala, either saredutant or osanetant, but not GR-205171, significantly elevated NGF contents (Fig. 4). As previously reported, NGF plays an essential role in the regeneration, maintenance, and development of the olfactory system of mammals (Miwa et al., 2002). In addition, NGF facilitates

Involvement of neurotrophin and CB systems in the mechanisms of action of NK receptor antagonists cholinergic neurotransmission between the nucleus basalis and amygdala which is important for cognitive functions (Moises et al., 1995). As a whole, it appears that the enhancement of brain regional levels of NGF constitutes an essential part of the biochemical alterations induced by certain NK antagonists. According to the role of the eCBs in the regulation of mood (Bambico et al., 2009) and a possible interaction between the eCBs and neurotrophin signaling (Aso et al., 2008; Williams et al., 2003), we investigated the possible involvement of the eCBs in the mechanisms of action of NK antagonists including the regulation of NGF production in the brain. The CB1 receptor neutral antagonist AM4113 by blocking the endogenous cannabinoid activity prevented the NK antagonists-induced enhancement of NGF levels in a dose-dependent fashion (Fig. 5C), while, AM4113 had no effect by itself (Fig. 6). These findings argue for the CB1-mediated up-regulation of brain NGF levels by the NK receptor antagonists. We also found that chronic exposure to the NK receptor antagonists (10 mg/kg) caused a significant elevation of the endocannabinoids, AEA and 2-AG, in distinct brain regions implicated in the regulation of emotional behavior and synaptic plasticity (Table 5). This, was prevented by the pharmacological CB1 receptor blockade in a dose-dependent fashion (Table 6C). These findings suggest an existence of the intrinsic eCB activity which may contribute to the mechanisms of action of the NK receptor antagonists and agree with previous data suggesting a pivotal role for the eCBs in the regulation of emotional states and neuroplasticity (Marsicano and Lutz, 2006; Viveros et al., 2007). As previously reported, pharmacological facilitation of the eCBs produces an antidepressant response and elevates the effects of antidepressants (Hill et al., 2006, 2008). Furthermore, the cannabinoid agonists or eCB enhancers have been shown to possess neuroprotective properties (Panikashvili et al., 2001; Zhuang et al., 2005) and promote hippocampal neurogenesis (Viveros et al., 2007) leading to the anxiolytic- and anti-depressant-like effects. As a whole, brain regional distribution of endocannabinoids following treatment with NK receptor antagonists suggests that cannabinoid system may be integral to the development and maintenance of effective coping strategies and emotional responses. Meanwhile, further investigation is required to find out more about the additional mechanism/s and signal transduction pathways linking the NK antagonists to endocannabinoids. Interestingly, in most cases, the eCB contents were increased within the brain regions in which the NK antagonists were also able to elevate NGF production (Fig. 4, Table 5). This may be in agreement with previous reports indicating an interaction between the neurotrophin and eCB systems (Aso et al., 2008; Williams et al., 2003). It appears that the regulatory effects of the NK antagonists on NGF levels depends on the integrity of the eCBs in certain brain regions and the CB1 receptors play a critical role in this regard. In conclusion, we demonstrated for the first time that the selective NK receptor antagonists elevate NGF levels in distinct brain regions relevant to the regulation of mood. Our data also provide evidence that the eCBs, especially through CB1 signaling, plays a pivotal role in the action of NK antagonists including the upregulation of NGF synthesis in gerbil brain. These findings may present an impetus for a better understanding of the pathophysiological mechanisms underlying neuropsychiatric disorders and suggest that the NK system could be a promising target for novel drug development.

915

Role of the funding source This study was funded in part by a grant (A-1156) from AJA University of Medical Sciences. Meanwhile, AJA University had no further role in study design, in the collection, analysis or interpretation of data, in writing of the manuscript, or in the decision to submit the paper for publication.

Contributors The corresponding author, Parichehr Hassanzadeh, designed the study, wrote the protocol, undertook the statistical analysis, and wrote the first draft of the manuscript. Author Anna Hassanzadeh participated in the performance of animal experiments and analysis of the results. Both authors managed the literature searches and analysis, contributed to and approved the final manuscript.

Conflict of interest Authors have no conflict of interest to declare.

Acknowledgements We would like to thank Dr. Ali Mahdavi, Department of Immunology, Tarbiat Modarres University, Tehran, Amir Farahbakhsh, Department of Molecular biology, Azad University, Parand, and Fatemeh Eini, AJA University of Medical Sciences, for their technical assistance. We gratefully acknowledge Dr. Alireza Khoshdel for proof-reading of the manuscript.

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