- Email: [email protected]
Fluvoxamine maleate effects on dopamine signaling in the prefrontal cortex of stressed Parkinsonian rats: Implications for learning and memory
Brain Research Bulletin 132 (2017) 75–81
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Fluvoxamine maleate effects on dopamine signaling in the prefrontal cortex of stressed Parkinsonian rats: Implications for learning and memory Ernest Dalléa, Willie M.U. Danielsb, Musa V. Mabandlaa, a b
MARK
⁎
School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban 4000, South Africa School of Physiology, University of the Witwatersrand, Johannesburg, South Africa
A R T I C L E I N F O
A B S T R A C T
Keywords: Prefrontal cortex Cognitive impairment Dopamine Serotonin Parkinson’s disease
Parkinson’s disease (PD) is also associated with cognitive impairment and reduced extrinsic supply of dopamine (DA) to the prefrontal cortex (PFC). In the present study, we looked at whether exposure to early life stress reduces DA and serotonin (5-HT) concentration in the PFC thus leading to enhanced cognitive impairment in a Parkinsonian rat model. Maternal separation was the stressor used to develop an animal model for early life stress that has chronic effects on brain and behavior. Sprague-Dawley rats were treated with the antidepressant Fluvoxamine maleate (FM) prior to a unilateral 6-hydroxydopamine (6-OHDA) lesion to model motor deficits in rats. The Morris water maze (MWM) and the forelimb use asymmetry (cylinder) tests were used to assess learning and memory impairment and motor deficits respectively. Blood plasma was used to measure corticosterone concentration and prefrontal tissue was collected for lipid peroxidation, DA, and 5-HT analysis. Our results show that animals exposed to early life stress displayed learning and memory impairment as well as elevated basal plasma corticosterone concentration which were attenuated by treatment with FM. A 6-OHDA lesion effect was evidenced by impairment in the cylinder test as well as decreased DA and 5-HT concentration in the PFC. These effects were attenuated by FM treatment resulting in higher DA concentration in the PFC of treated animals than in non-treated animals. This study suggests that DA and 5-HT signaling in the PFC are responsive to FM and may reduce stress-induced cognitive impairment in PD.
1. Introduction Parkinson’s disease (PD) is a neurodegenerative condition in which dementia or cognitive dysfunction may precede the motor dysfunction that follows midbrain dopaminergic neuron loss (Narayanan et al., 2013). The prevalence of cognitive dysfunction is high with reports indicating this non-motor symptom to negatively affect the quality of life of about 60% of patients with PD and to cause morbidity and mortality in 36% of patients at the early onset of the disease (Cools et al., 2002; Foltynie et al., 2004; Forsaa et al., 2010; Solari et al., 2013). Patients suffering from PD, therefore, appear to be particularly susceptible to develop cognitive impairments with rates of 4–6 times that of normal aging being recorded (Aarsland et al., 2005; Narayanan et al., 2013). More importantly, studies have shown that cognitive impairment may occur at an early stage of PD and this seems to correlate well with dopaminergic dysfunction in the prefrontal cortex (PFC) (Brück et al., 2005; Chudasama and Robbins, 2006). However, to date, the cellular mechanism underlying the development of cognitive impairment in PD remains largely unknown.
⁎
Corresponding author. E-mail address: [email protected] (M.V. Mabandla).
http://dx.doi.org/10.1016/j.brainresbull.2017.05.014 Received 21 February 2017; Received in revised form 9 May 2017; Accepted 22 May 2017 Available online 24 May 2017 0361-9230/ © 2017 Published by Elsevier Inc.
The PFC is known to play a key role in short and long-term learning and memory processes (Takashima et al., 2006; Corcoran and Quirk, 2007). Deficits in cognitive planning and spatial working memory that occur in PD and progressively increase in intensity with time, have been associated with damage of the PFC (Cools et al., 2002; Solari et al., 2013). During the early stages (non-motor symptoms) of PD, cognitive impairment may correlate with dopaminergic dysfunction in the PFC, while during advanced stages (motor symptoms) of PD, the direct dopaminergic projection from the ventral tegmental area to the frontal cortex is affected resulting in cortical dopamine (DA) depletion (JavoyAgid and Agid, 1980; Scatton et al., 1983; Lupien et al., 2009; De la Fuente-Fernandez, 2012; Yuen et al., 2012; Solari et al., 2013). The PFC area of the brain receives a broad range of sensory and limbic inputs critical for appropriate behavioral task execution. However, altered DA transmission within the PFC results in abnormal information processing, leading to learning and memory deficits (Solari et al., 2013). In this regard, it has been suggested that learning and memory impairment may predict the development of dementia in PD that is so highly prevalent across the entire course of the disease (Aarsland et al., 2005;
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
2.2. Drugs and reagents
Aarsland and Kurz, 2010). We have recently shown that the antidepressant Fluvoxamine maleate (FM) is not only useful in treating major anxiety/depressivelike symptoms but also attenuates neurotoxin induced-DA degeneration in an animal model for PD (Dallé et al., 2017). The effects of antidepressants on serotonergic and dopaminergic transmission are well established (Tao et al., 2016). These effects include increasing serotonin (5-HT) and DA levels in the striatum and the PFC (Lupien et al., 2009; Hemmerle et al., 2012). More importantly, the effects of antidepressants on cognition are widely known since long-lasting cognitive deficits induced by stress can be attenuated by some antidepressant (Nikiforuk and Popik, 2011). However, finding the mechanism of cognitive dysfunction in PD is critical for treating cognitive symptoms of PD, since to date, only few effective treatments are available. Moreover, to the best of our knowledge, apart from Levodopa that has been shown to improve cognitive symptoms of PD depending on the stage of the disease and the integrity of striatal DA signaling, no studies have looked at the effects of an antidepressant to treat PD-induced DA depletion in the PFC (Kulisevsky et al., 2000; Cools et al., 2002; Muller et al., 2001; Cools, 2006; Pascual-Sedano et al., 2008). We hypothesized that chronic treatment with FM will reduce learning and memory deficits and counteracts 6-OHDA-induced Parkinsonian symptoms in stressed rats. The present study consequently aimed to investigate the effects of FM treatment on cognitive deficits in a Parkinsonian rat model and whether serotonergic and/or dopaminergic mechanisms of FM in the PFC could contribute to an antiparkinsonian effect in stressed rats.
The drug Fluvoxamine maleate, Temgesic and Biotane were obtained from Pharmed Pharmaceuticals LTD (Rochdale Park, Durban, South Africa). Desipramine (D3900), atropine, pentobarbital and 6OHDA were purchased from Sigma (St. Louis MO, USA). The Corticosterone (RE52211), Dopamine (RE59161) and Serotonin (RE59121) ELISA kits were purchased from IBL International GmbH (Hamburg, Germany). The lipid peroxidation (MDA) assay kit (K739100) was obtained from BioVision (Mountain view, CA, USA). 2.3. Maternal separation Maternal separation was the stressor used to enhance learning and memory deficits (non-motor symptoms of PD) as per study by Lupien et al. (2009) and Hermmerle et al. (2012). This protocol was also used so as to investigate whether addressing these symptoms at their early stage with FM could delay the motor symptoms in a model of PD. The maternal separation stress protocol was carried out once a day from PND 2 to PND 14. We used a stress protocol previously described in Mabandla and Russell (2010) and Dalle et al. (2016). Briefly, the pups were taken away from their dams and kept in a separate room for 3 h (09:00–12:00). All normally reared pups were left undisturbed with their dams. 2.4. Behavioral tests Behavioral tests were used to assess the effects of FM on learning and memory in a maternally separated rat as well as to assess the effects of the drug on motor dysfunction in a Parkinsonian rat model. The behavioral tests used included the MWM test and the limb-use asymmetry test (cylinder test). The tests were performed pre- as well as post-lesion with 6-OHDA. All behavioral tests were video-recorded for subsequent scoring and manually analyzed by an evaluator blind to the study.
2. Materials and methods 2.1. Animals The experimental protocol used in this study was reviewed and approved by the Animal Research Ethics Committee of the University of KwaZulu-Natal (018/15/Animal) in accordance with the guidelines of the National Institutes of Health, USA. The sample size was set according to previous studies where the statistical power was shown (Eng, 2003; Dalle et al., 2016). A total of 60 male Sprague-Dawley rats obtained from the Biomedical Resource Unit of the University of KwaZulu-Natal were used in this study. They were housed in polypropylene cages (38 × 32 × 16 cm) under controlled temperature (21 ± 2 °C) and humidity (55–60%). Food and water were freely available. The daily light/dark cycle was 07:00–19:00 (Mabandla and Russell, 2010). On post-natal day (PND) 1, the rats were sexed and culled to 6 male pups per litter and randomly divided into 6 equal groups as follows: non-stressed (NS), non-stressed treated with saline (NSS), non-stressed treated with FM (NSF), maternally separated (MS), maternally separated treated with saline (MSS) and maternally separated treated with FM (MSF). The rats were weaned on PND 21 after which they were kept 6 per cage (Dalle et al., 2016). On PND 29, all treated groups received saline or FM intraperitoneally from PND 29–59 once daily. NSS and MSS rats received vehicle injections (saline 1 ml/ 250 g of body weight) and were lesioned with saline to control for the effects of handling, stress, lesion and the injection itself. NSS and MSS data were not included since no significant effect was found between saline treated rats and non-treated rats. Lesion refers to the intracerebral injection of the neurotoxin 6-OHDA on PND 60 in all groups. The animals were weighed prior to all experimental procedures and were brought to the experimental room at least 1 h before experimentation. The learning and memory ability test was assessed on PND 28, 58 and 74 using the Morris water maze (MWM) apparatus (Vorhees and Williams, 2006; Beraki et al., 2009). Limb-use asymmetry was assessed on PND 59 and 75 using the cylinder test (Mabandla and Russell, 2010). The rats were sacrificed on PND 76. All experimental procedures were conducted between 09:00 and 16:00.
2.4.1. Morris water maze (MWM) test The MWM test was used to assess spatial learning and memory (D’hooge and De Deyn, 2001). The MWM apparatus used was a circular tank (1 m in diameter) that is divided into 4 quadrants filled with water (22–23° C) in where a hidden square plexiglass platform (10 × 10 cm wide and 20 cm high) is submerged (1 mm) bellow the water surface. The hidden platform was placed in one of the quadrants and each quadrant had a visual cue to help the rat in its navigation. All animals were exposed to training sessions (4 trials per day) over consecutive days (PND 25–27) (Vorhees and Williams, 2006). The latency time (time taken to reach the hidden platform) was recorded and was taken as the learning process (Morris, 1984). A probe test was conducted (without the platform) to assess the ability of the rats to remember the quadrant in which the platform was located (Garthe and Kempermann, 2013). 2.4.2. The limb-use asymmetry test (Cylinder test) The cylinder test was used to assess the forelimb used during exploratory activity in the plexiglass cylinder and for landing over a period of 5 min. As the neurotoxin 6-OHDA was injected into the left hemisphere, a successful model of Parkinsonism would result in a bias towards left forelimb use after lesion (Tillerson et al., 2001; Mabandla and Russell, 2010). The cylinder was made of transparent plexiglass (20 cm in diameter and 30 cm in height). The rats were assessed for percentage limb-use of the impaired (right) limb by using the following equation: % limb use of impaired = [(impaired + ½ both)/(impaired + unimpaired + both)] × 100. Both, refers to the use of both the impaired and unimpaired limbs during exploratory activity (Tillerson et al., 2001). 76
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
300 μl of the Malondialdehyde (MDA) lysis buffer was homogenized on ice using a sonicator (CML-4, Fisher, USA) before being centrifuged at 13000 rpm for 10 min. The supernatant (200 μl) from each homogenized sample was pipetted into a microcentrifuge tube. To prepare the standards, 10 μl of MDA standard was diluted with 407 μl of bidistilled water so as to prepare a solution of 0.1 M of MDA. 0.1 M MDA (20 μl) solution was diluted with bidistilled water (980 μl) resulting in a 2 mM MDA standard. Thiobarbituric acid (TBA) solution (600 μl) was then added to the vials containing the Standards and samples. This was followed by incubation for 1 h in a water bath at 95° C. Vials containing the Standards as well as the control samples were allowed to cool at room temperature for 10 min after which 200 μl of each solution (Standards and samples) was pipetted in duplicate into a 96-well microplate. Absorbance was read at 532 nm using a spectrophotometer (Spectrostar Nano BMG LABTECH, Germany).
2.5. Fluvoxamine maleate (FM) treatment Prior to treatment, the rats were weighed and the volume of FM to be injected (1 ml/250 g of body weight) was calculated accordingly. FM was always freshly prepared by dissolving in saline after which it was intraperitoneally (i.p) injected (25 mg/kg) once daily during the treatment period (Gerstenberg et al., 2003; Muck-Seler et al., 2012; Dalle et al., 2016). 2.6. Hemiparkinsonian rat model (6-OHDA lesion) 6-OHDA was injected stereotaxically into the medial forebrain bundle (MFB) as per Paxinos and Watson, (1998). The norepinephrine reuptake blocker desipramine HCl (15 mg/kg, i.p.) was injected 30 min prior to 6-OHDA infusion in order to inhibit 6-OHDA uptake by noradrenergic neurons (Goodman et al., 1990). Before 6-OHDA infusion, the rats were anaesthetized with sodium pentobarbital (60 mg/kg, i.p.) followed by an atropine (0.2 mg/kg, i.p.) injection so as to facilitate respiration while the rat was unconscious. This was followed by shaving the head before positioning it in the stereotaxic apparatus (David Kopf Instruments, Tujunga CA, USA). The skin covering the scalp was disinfected with Biotane and a midline incision was made to expose the skull. A burr hole was drilled on the skull at the following coordinates: anterior-posterior (AP) = +4.7 mm anterior to lambda; medio-lateral (ML) = +1.6 mm from midline suture (Paxinos and Watson, 1998). At these coordinates, a Hamilton syringe was lowered 8.4 mm ventral to dura before 6-OHDA HCl (5 μg/4 μl) dissolved in 0.2% ascorbic acid was injected into the left MFB over a period of 8 min. The needle was kept in the MFB for 1 min prior to the injection and for 2 min following the injection so as to maximize diffusion of the neurotoxin. After the needle was retracted, the incision was sutured and cleaned and the rat was kept warm under a heating lamp so as to prevent hypothermia during recovery. The rats were injected with the analgesic Temgesic (0.05 mg/kg subcutaneously) before being returned to their home cages.
2.7.2.2. Dopamine (DA) in the PFC. Dissected PFC tissue was weighed and placed in Eppendorf tubes prior to freezing in liquid nitrogen before being stored at −80 °C in a bio-freezer until the day of analysis. On the day of analysis, the PFC tissue (n = 6/group) was removed from the bio-freezer and allowed to thaw at room temperature. EDTA–HCL buffer (500 μl) was added to each tube containing PFC tissue. The tissue was homogenized in a sonicator before being centrifuged at 3500 rpm for 10 min at 4 °C. The supernatant was pipetted into new Eppendorf tubes for DA analysis using a dopamine ELISA kit according to the manufacturer’s protocol. The DA analysis protocol consisted of an extraction procedure that was followed by quantification. Both steps were conducted on the same day. Briefly, extraction consisted of pipetting 20 μl of each standard, 20 μl of each control and 500 μl of each sample into respective wells in the extraction plate. The standards and controls were diluted with bidistilled water in order to correct for differences in volume as per the manufacturer’s instructions. For the quantification procedure, the extracted standards and controls were diluted with 500 μl of release buffer. Pre-diluted standards, controls and 100 μl of each extracted sample (without pre-dilution) were pipetted into respective wells containing COMT Enzyme solution (75 μl) supplied with the kit. All samples, standards and controls were analysed in duplicate. The optical density of the samples was measured using a microtiter plate reader (SPECTROstar Nano, BMG LABTECH GmbH, Ortenberg, Germany) at 405 nm, within 10 min adding the stop solution.
2.7. Decapitation and neurochemistry On PND 76 (16 days post-lesion with 6-OHDA), the rats were decapitated using a sharp guillotine. Trunk blood and PFC tissue were collected. Trunk blood was collected in EDTA coated test tubes and immediately centrifuged using a refrigerated centrifuge (Labofurge 200, Heraeus Sepatech, Hanau, Germany). Plasma was transferred into Eppendorf tubes and snap frozen in liquid nitrogen before being stored in a bio-freezer at −80 °C until the day of analysis. Plasma corticosterone concentration (n = 10/group) was analyzed using a corticosterone ELISA kit according to the manufacturer’s protocol.
2.7.2.3. Serotonin (5-HT) in the PFC. Hydrochloric acid (0.05 M) mixed with 0.1% ascorbic acid was added to each sample (n = 6/group), homogenized with a sonicator then shaken (protected from sun light) for 1 h before being centrifuged for 20 min at 4000 rpm. The supernatant was collected into new Eppendorf tubes. Standards, controls, and samples (25 μl each) were pipetted into reaction tubes provided with the kit. Acylation Buffer (500 μl) and Acylation Reagent (25 μl) were added to all tubes, mixed and incubated for 15 min at room temperature. The acylated standards, controls, and samples (25 μl each) were pipetted into respective wells of the 5-HT microtiter strips (provided with the kit). 5-HT antiserum (100 μl) was pipetted into each well, incubated for 30 min after which each well was washed with 300 μl wash buffer and then dried. Thereafter, 100 μl of Conjugate solution was pipetted into each well, allowed to incubate for 15 min. The contents were then discarded after which the well was washed and dried. A volume of 100 μl of Substrate solution was pipetted into each well and allowed to incubate for 15 min before the Stop solution (100 μl) was added. All samples, standards, and controls were read in duplicate. The absorbance of the solution in the wells was read within 10 min using a microplate reader set at 450 nm.
2.7.1. Plasma corticosterone concentration On the day of analysis, blood plasma samples were allowed to thaw on ice. A volume of 20 μl of each standard, control, and plasma samples was dispensed into the appropriate wells of a 96 well plate. Enzyme conjugate (200 μl) was added to each well, mixed and incubated for 1 h at room temperature. Following the addition of Substrate solution (100 μl) to each well, the plate was incubated at room temperature for 15 min. Following incubation, Stop solution (50 μl) was added after which the optical density of the standards, controls, and samples was measured using a microtiter plate reader (SPECTROstar Nano, BMG LABTECH GmbH, Ortenberg, Germany) at 450 nm within 10 min as per the manufacturer’s protocol. 2.7.2. Brain tissue 2.7.2.1. Lipid peroxidation. Lipid peroxidation (Malondialdehyde) quantification as a measure of oxidative stress was evaluated in the PFC of all groups (n = 6/group) using the method described in Hall and Bosken (2009) and the manufacturer’s protocol. Briefly, PFC tissue in 77
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
2.8. Statistical analysis All results are presented as mean ± SEM. The data was analyzed using the software program GraphPad Prism (version 5.0, San Diego, California, USA). Data normality was assessed by the KolmogorovSmirnov test and where data met requirements, parametric or nonparametric tests were used. The main factors in each analysis included stress and treatment. When measuring only one variable (stress versus non-stressed), a paired t-test was used for analysis. Two-way analysis of variance (ANOVA) was used for behavior as well as corticosterone, lipid peroxidation, DA and 5-HT concentration data analysis. In each analysis, significant main effects were followed by Bonferroni post hoc test. p < 0.05 was considered significant in all analysis. 3. Results Fig. 1. Time taken by maternally separated and non-stressed rats to find the hidden platform in the MWM on the training days (PND 25, 26 and 27) and on the test day (PND 28). **(Day 1 vs. Day 2, p = 0.0038), ***(Day 2 vs. Day 3, p = 0.0001), and ***(Day 1 vs. Day 3, p < 0.0001), Paired t-tests in non-stressed rats. *(Day 1 vs. Day 2, p = 0.0149) and ***(Day 1 vs. Day 3, p = 0.0001). Paired t-tests in maternally separated rats. *(NS Day 3 vs. MS Day 3, p = 0.0107, Paired t tests). Data are presented as mean ± SEM. (n = 20/group). On PND 28: ***(NS vs. MS, p = 0.0001, paired t-test). Data are presented as mean ± SEM. (n = 20/group). Effects of FM in the memory ability of maternally separated and non-stressed rats with regards to the time spent in the quadrant of the hidden platform in the MWM on PND 59. *(NS vs. MS, p = 0.0107, paired t-test), [*(NS vs. NSF), F (1, 36) = 8.963, p = 0.050 and **(MS vs. MSF), F(1, 36) = 11.16, p = 0.0020, two-way ANOVA with Bonferroni post hoc test]. Data are presented as mean ± SEM. (n = 10/group). Effects of FM in the memory ability of maternally separated and non-stressed rats with regards to the time spent in the quadrant of the hidden platform in the MWM on PND 74. ***(NS vs. MS, p < 0.0001, paired ttest), and [***(MS vs. MSF), F(1, 36) = 14.88, p = 0.0005, two-way ANOVA with Bonferroni post hoc test]. Data are presented as mean ± SEM. (n = 10/group).
3.1. Morris water maze (MWM) test Training sessions on the MWM took place on PND 25–27. A training effect was present in the non-stressed groups **(Day 1 vs. Day 2 p = 0.0038), ***(Day 2 vs. Day 3, p = 0.0001) and ***(Day 1 vs. Day 3, p < 0.0001). A similar effect was present in the stressed groups *(Day 1 vs. Day 2, p = 0.0149) and ***(Day 1 vs. Day 3, p = 0.0001). Overall, non-stressed rats had a shorter latency on day 3 *(NS Day 3 vs. MS Day 3, p = 0.0107). The effects of maternal separation on short and long-term learning and memory were assessed on PND 28, 58 and 74. On PND 28 (pretreatment stage), there was a stress effect as maternally separated rats took longer to find the platform ***(NS vs. MS, p = 0.0001). On PND 58, the probe test showed that there was a stress effect as maternally separated rats struggled to find the quadrant with the hidden platform *(NS vs. MS, p = 0.0107). However, there was a treatment effect as FM-treated rats spent more time in the quadrant with the hidden platform in both non-stressed and maternally separated rats [*(NS vs. NSF), F (1, 36) = 8.963, p = 0.050 and **(MS vs. MSF), F(1, 36) = 11.16, p = 0.0020]. Similarly, on PND 74, there was a stress effect as maternally separated rats spent less time in the quadrant of the hidden platform when compared to non-stressed rats *(NS vs. MS, p = 0.0171). A treatment effect on memory was observed as FM-treated rats spent more time in the quadrant of the hidden platform in maternally separated rats [*(MS vs. MSF), F(1, 36) = 3.868, p = 0.0333]. Data shown in Fig. 1. 3.2. Cylinder test Forelimb use asymmetry was assessed on PND 58 and PND 75. Prior to 6-OHDA lesion (PND 58), no asymmetry was observed in all the rats (Data not shown). On PND 75, a stress effect was evident as 6-OHDA lesion in these rats resulted in decreased use of the right (impaired) forelimb when touching the wall of the cylinder *(NS vs MS, p = 0.0199). A treatment effect was present as FM treatment reduced forelimb use asymmetry caused by the 6-OHDA lesion in both the nonstressed and maternally separated rats [*(NS vs. NSF, F(1, 36) = 6.196, p = 0.0176) and **(MS vs. MSF), F(1, 36) = 16.65, p = 0.0002]. In addition, significant main effects of stress and FM treatment with respect to the use of the right (impaired) forelimb for landing was observed *(NS vs MS, p = 0.0269), [**(NS vs. NSF), F(1, 36) = 12.59, p = 0.0011] and **(MS vs. MSF), F(3, 36) = 7.708, p = 0.0022]. Data presented in Fig. 2.
Fig. 2. Number of times the rat used the impaired (right) forelimb to touch the wall of the cylinder and to land on the floor expressed as a percentage of the total number of times it touched the wall of the cylinder or landed on the floor of the cylinder on PND 75. All groups were lesioned with 6-OHDA on PND 60. Wall touch, *(NS vs MS, p = 0.0199, paired t-test), [*(NS vs. NSF, F(1, 36) = 6.196, p = 0.0176) and **(MS vs. MSF), F(1, 36) = 16.65, p = 0.0002, two-way ANOVA with Bonferroni post hoc test]. Landing on the floor, *(NS vs MS, p = 0.0269, paired t-test), [**(NS vs. NSF), F(1, 36) = 12.59, p = 0.0011] and ***(MS vs. MSF), F(3, 36) = 7.708, p = 0.0022, two-way ANOVA with Bonferroni post hoc test]. Values are expressed as mean ± SEM (n = 10/group).
elevated than those of the non-stressed rats ***(NS vs. MS, p = 0.0002). A treatment effect was found as FM treatment significantly decreased plasma corticosterone levels in maternally separated rats [**(MS vs. MSF), F(1, 36) = 9.84, p = 0.0039]. In addition, as one of the principal mechanism of neuronal injury associated with oxygen radicals in PD, lipid peroxidation levels were measured to quantify oxidative stress and to evaluate the antioxidant capacity of FM. A stress effect was observed ***(NS vs. MS, p = 0.0007). Treatment with FM
3.3. Corticosterone and lipid peroxidation concentration Plasma corticosterone and Lipid peroxidation concentration were measured on PND 76. We found a stress effect as the plasma corticosterone levels of maternally separated rats was significantly 78
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
enhanced cognitive impairment in a Parkinsonian rat model. The MWM test showed that exposure to early life stress resulted in learning and memory deficits. We also found that exposure to early life stress exacerbated the effects of 6-OHDA on motor function (Mpofana et al., 2014; Mabandla and Russell, 2010; Dalle et al., 2016). These results are in agreement with previous studies and provide evidence that exposure to maternal separation impairs learning and memory (Solari et al., 2013; Aisa et al., 2007; Pienaar et al., 2008; Tata et al., 2015). However, treatment with the antidepressant FM improved learning and memory impairment in both the non-stressed and stressed groups. Although this finding is the first to find FM’s therapeutic effect in relieving cognitive impairment in a rat model of PD, there is evidence suggesting that impaired learning and memory could be the result of elevated glucocorticoid (corticosterone) levels (Fann et al., 2001; Nowakowska et al., 2001; Harmer et al., 2002; Yau et al., 2002). Studies have shown that early maternal separation disturbs the hypothalamic-pituitary-adrenal (HPA) axis and leaves the brain more vulnerable to subsequent traumatic events later in life (Lupien et al., 2009; Mpofana et al., 2014). Reducing the cumulative lifetime exposure to corticosterone excess by long-term exposure to FM may have prevented the emergence of cognitive deficits in our rats. We, therefore, speculate that deficits may have occurred as a result of malfunctioning corticosterone regulation which is known to have a critical role in the development of cognitive function (Duman et al., 2000; Song et al., 2006). In the cylinder test, as expected, our results showed a preference to using the unimpaired forelimb in both groups (NS and MS) following 6OHDA lesion. However, exposure to early life stress exacerbated this asymmetry. These results are in agreement with previous findings that used the same animal model (Mabandla and Russell, 2010; Mpofana et al., 2014; Dalle et al., 2016). We further found that treatment with FM reduced this asymmetrical use of the forelimbs suggesting that FM treatment may be neuroprotective against DA degeneration in the nigrostriatal pathway. We have recently shown that FM possesses antiinflammatory and antioxidant properties (Dallé et al., 2017). Although further specific investigations need to be done, these properties may have been essential in ameliorating the toxic effects of 6-OHDA in NSF and MSF rats through reinforcement of enzymatic anti-inflammatory and antioxidant defenses (Mabandla et al., 2015). Plasma corticosterone concentration was measured to evaluate the effectiveness of our early life model for stress. Our results did indeed show that we have a stressed animal model with basal corticosterone concentration being higher in the maternally separated rats (Daniels et al., 2004). However, there was a reduction in plasma corticosterone concentration in the stressed rats following treatment with FM. It has been shown that FM can activate the HPA axis resulting in neuroendocrine responses via specific serotonergic neurons (Yamauchi et al., 2006). As learning and memory deficits as well as increased corticosterone concentration have been linked to anxiety-like behavior, FM’s ability to decrease corticosterone concentration in maternally separated rats confirms the anxiolytic effect of the drug as shown by Hiemke and Hartter (2000) and Irons (2005). Studies have shown that exposure to stress induces lipid peroxidation in the brain (Liu et al., 1994; Matsumoto et al., 1999; Madrigal et al., 2001). The positive correlation between plasma corticosterone concentration and lipid peroxidation suggests that elevated corticosterone concentration plays a role in increasing lipid peroxidation (Sahin and Gumuslu, 2004). We found that exposure to stress resulted in increased lipid peroxidation. However, FM treatment resulted in decreased lipid peroxidation levels in the PFC of stressed rats. These results are in agreement with results from previous studies that showed that antidepressants decreased oxidative stress in the PFC of stressed animals (Cline et al., 2015). We, therefore, suggest that the antioxidant effects of FM may be responsible for mopping up the free radicals generated by the autooxidation of 6-OHDA. In PD, reduction of cortical dopaminergic transmission can be
Fig. 3. Effect of FM on plasma corticosterone concentration in 6-OHDA lesioned, maternally separated (MS, MSF) and non-stressed (NS, NSF) rats on PND 76. ***(NS vs. MS, p = 0.0002, paired t-test) and [**(MS vs. MSF), F(1, 36) = 9.84, p = 0.0039, twoway ANOVA with Bonferroni post hoc test]. Data presented as mean ± SEM. (n = 10/ group). Effect of FM on lipid peroxidation levels in 6-OHDA lesioned, maternally separated (MS, MSF) and non-stressed (NS, NSF) rats on PND 76. ***(NS vs. MS, p = 0.0007, paired t-test) and [**(MS vs. MSF), F(1, 20) = 14.43, p = 0.0011, two-way ANOVA with Bonferroni post hoc test]. Data presented as mean ± SEM. (n = 6/group).
significantly decreased lipid peroxidation levels in maternally separated rats [**(MS vs. MSF), F(1, 20) = 14.43, p = 0.0011]. Data presented in Fig. 3. 3.4. Dopamine (DA) and serotonin (5-HT) concentration DA and 5-HT concentration were measured on PND 76. A stress effect that resulted in decreased DA concentration in the maternally separated rats was observed *(NS vs. MS, p = 0.0385). Treatment with FM attenuated the decrease in DA concentration in maternally separated rats [*(MS vs. MSF), F(1, 20) = 6.627, p = 0.0181]. In addition, a stress effect resulting in decreased 5-HT concentration was observed *(NS vs. MS, p = 0.0219). Treatment with FM affected 5-HT transmission in non-stressed rats [**(NS vs. NSF), F(1, 20) = 11.21, p = 0.0032]. Data presented in Fig. 4. 4. Discussion In the present study, we looked at whether exposure to early life stress exacerbates DA and 5-HT depletion in the PFC thus leading to
Fig. 4. Effect of FM on DA concentration in 6-OHDA lesioned, maternally separated (MS, MSF) and non-stressed (NS, NSF) rats on PND 76. *(NS vs. MS, p = 0.0385, paired t-test) and [*(MS vs. MSF), F(1, 20) = 6.627, p = 0.0181, two-way ANOVA with Bonferroni post hoc test]. Data presented as mean ± SEM. (n = 6/group). Effect of FM on 5-HT concentration in 6-OHDA lesioned, maternally separated (MS, MSF) and non-stressed (NS, NSF) rats on PND 76. *(NS vs. MS, p = 0.0219, paired t-test) and [**(NS vs. NSF), F(1, 20) = 11.21, p = 0.0032, two-way ANOVA with Bonferroni post hoc test]. Data presented as mean ± SEM. (n = 6/group).
79
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
Parkinson’s disease. Brain Pathol. 20, 633–639. Aarsland, D., Zaccai, J., Brayne, C., 2005. A systematic review of prevalence studies of dementia in Parkinson’s disease. Mov. Disord. 20, 1255–1263. Aisa, B., Tordera, R., Lasheras, B., Del Rio, J., Ramirez, M.J., 2007. Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats. Psychoneuroendocrinology 32, 256–266. Arnsten, A.F., Raskind, M.A., Taylor, F.B., Connor, D.F., 2015. The effects of stress exposure on prefrontal cortex: translating basic research into successful treatments for post-traumatic stress disorder. Neurobiol. Stress 1, 89–99. Belujon, P., Grace, A.A., 2015. Regulation of dopamine system responsivity and its adaptive and pathological response to stress. Proc. R. Soc. B: Biol. Sci. 282. Beraki, S., Diaz-Heijtz, R., Tai, F., Ogren, S.O., 2009. Effects of repeated treatment of phencyclidine on cognition and gene expression in C57BL/6 mice. Int. J. Neuropsychopharmacol. 12, 243–255. Brück, A., Aalto, S., Nurmi, E., Bergman, J., Rinne, J.O., 2005. Cortical 6- [18F]fluoro-ldopa uptake and frontal cognitive functions in early Parkinson’s disease. Neurobiol. Aging 26, 891–898. Buoli, M., Cumerlato Melter, C., Caldiroli, A., Altamura, A.C., 2015. Are antidepressants equally effective in the long-term treatment of major depressive disorder? Hum. Psychopharmacol. 30, 21–27. Chudasama, Y., Robbins, T.W., 2006. Functions of frontostriatal systems in cognition: comparative neuropsychopharmacological studies in rats monkeys and humans. Biol. Psychol. 73, 19–38. Cline, B.H., Anthony, D.C., Lysko, A., Dolgov, O., Anokhin, K., Schroeter, C., Malin, D., Kubatiev, A., Steinbusch, H.W., Lesch, K.P., Strekalova, T., 2015. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav. Brain Res. 276, 118–129. Cools, R., 2006. Dopaminergic modulation of cognitive function-implications for L-DOPA treatment in Parkinson’s disease. Neurosci. Biobehav. Rev. 30, 1–23. Cools, R., Stefanova, E., Barker, R.A., Robbins, T.W., Owen, A.M., 2002. Dopaminergic modulation of high-level cognition in Parkinson’s disease: the role of the prefrontal cortex revealed by PET. Brain 125, 584–594. Corcoran, K.A., Quirk, G.J., 2007. Activity in prelimbic cortex is necessary for the expression of learned, but not innate, fears. J. Neurosci. 27, 840–844. D’hooge, R., De Deyn, P.P., 2001. Applications of the Morris water maze in the study of learning and memory. Brain Res. Brain Res. Rev. 36, 60–90. Dalle, E., Daniels, W.M., Mabandla, M.V., 2016. Anti-parkinsonian effects of fluvoxamine maleate in maternally separated rats. Int. J. Dev. Neurosci. 53, 26–34. Dallé, E., Daniels, W.M.U., Mabandla, M.V., 2017. Fluvoxamine maleate normalizes striatal neuronal inflammatory cytokine activity in a parkinsonian rat model associated with depression. Behav. Brain Res. 316, 189–196. Daniels, W.M., Pietersen, C.Y., Carstens, M.E., Stein, D.J., 2004. Maternal separation in rats leads to anxiety-like behavior and a blunted ACTH response and altered neurotransmitter levels in response to a subsequent stressor. Metab. Brain Dis. 19, 3–14. De la Fuente-Fernandez, R., 2012. Frontostriatal cognitive staging in Parkinson’s disease. Parkinson’s Dis. 561046. Duman, R.S., Malberg, J., Nakagawa, S., D’Sa, C., 2000. Neuronal plasticity and survival in mood disorders. Biol. Psychiatry 48, 732–739. Dymecki, J., Lechowicz, W., Bertrand, E., Szpak, G.M., 1996. Changes in dopaminergic neurons of the mesocorticolimbic system in Parkinson’s disease. Folia Neuropathol. 34, 102–106. Eng, J., 2003. Sample size estimation: how many individuals should be studied? Radiology 227, 309–313. Fann, J.R., Uomoto, J.M., Katon, W.J., 2001. Cognitive improvement with treatment of depression following mild traumatic brain injury. Psychosomatics 42, 48–54. Foltynie, T., Brayne, C.E., Robbins, T.W., Barker, R.A., 2004. The cognitive ability of an incident cohort of Parkinson’s patients in the UK. The CamPaIGN study. Brain 127, 550–560. Forsaa, E.B., Larsen, J.P., Wentzel-Larsen, T., Alves, G., 2010. What predicts mortality in Parkinson disease?: a prospective population-based long-term study. Neurology 75, 1270–1276. Garthe, A., Kempermann, G., 2013. An old test for new neurons: refining the Morris water maze to study the functional relevance of adult hippocampal neurogenesis. Front. Neurosci. 7, 63. Gerstenberg, G., Aoshima, T., Fukasawa, T., Yoshida, K., Takahashi, H., Higuchi, H., Murata, Y., Shimoyama, R., Ohkubo, T., Shimizu, T., Otani, K., 2003. Relationship between clinical effects of fluvoxamine and the steadystate plasma concentrations of fluvoxamine and its major metabolite fluvoxamino acid in Japanese depressed patients. Psychopharmacology 167, 443–448. Goodman, W.K., Price, L.H., Delgado, P.L., Palumbo, J., Krystal, J.H., Nagy, L.M., Rasmussen, S.A., Heninger, G.R., Charney, D.S., 1990. Specificity of serotonin reuptake inhibitors in the treatment of obsessive-compulsive disorder. Comparison of fluvoxamine and desipramine. Arch. Gen. Psychiatry 47, 577–585. Hall, E.D., Bosken, J.M., 2009. Measurement of oxygen radicals and lipid peroxidation in neural tissues. Curr. Protoc. Neurosci. 1–51 Chapter 7, Unit 7.17. Harmer, C.J., Bhagwagar, Z., Cowen, P.J., Goodwin, G.M., 2002. Acute administration of citalopram facilitates memory consolidation in healthy volunteers. Psychopharmacology (Berl.) 163, 106–110. Hiemke, C., Hartter, S., 2000. Pharmacokinetics of selective serotonin reuptake inhibitors. Pharmacol. Ther. 85, 11–28. Huot, P., Fox, S.H., Brotchie, J.M., 2015. Monoamine reuptake inhibitors in Parkinson’s disease. Parkinsons Dis. 2015, 609428. Irons, J., 2005. Fluvoxamine in the treatment of anxiety disorders. Neuropsychiatr. Dis. Treat. 1, 289–299. Javoy-Agid, F., Agid, Y., 1980. Is the mesocortical dopaminergic system involved in
associated with cognitive deficits (Scatton et al., 1982, 1983; Kulisevsky et al., 2000). Our results show that DA concentration was lower in the PFC of the stressed rats. This suggests that DA concentration is responsive to stress and may modulate its effects via the mesocortical pathway in agreement with previous studies that showed that stress alters DA metabolism in the PFC (Pani et al., 2000; Luo et al., 2014; Arnsten et al., 2015). The PFC plays a role in controlling the HPA axis’ responds to stress by modulating the neuroendocrine function via the mesocortical pathway (projections from the ventral tegmental area to the PFC) (Narayanan et al., 2012; Belujon and Grace, 2015). In fact, the PFC region receives DA projections from the ventral tegmental and medial nigral regions of the midbrain which may also degenerate in the course of PD (Javoy-Agid and Agid, 1980; Dymecki et al., 1996; Williams and Goldman-Rakic, 1998). Our results suggest that maternal separation resulted in suppression of the limbic DA transmission in stressed rats. However, FM treatment preserved DA transmission in the PFC of stressed rats suggesting a neuroprotective effect of the drug. Cools et al. (2002) reported that increases in DA transmission in the PFC contribute to cognitive stability in predicting the consequences of caudate nucleus dysfunction. Therefore, it is possible that similar to Levodopa, FM may have induced blood flow increases in the PFC regions so as to preserve cortical dopaminergic transmission. We also found that the concentration of 5-HT was lower in the stressed animals. These results agree with previous findings that showed that 6-OHDA lesioned maternally separated rats have altered 5-HT concentration in the PFC (Daniels et al., 2004; Sun et al., 2015). However, FM treatment did not affect 5-HT concentration in stressed animals. We speculated that serotonergic hyperinnervation may have occurred in NSF rats but not in MSF, and this may have compensated for the dopaminergic denervation (Maeda et al., 2003; Buoli et al., 2015; Huot et al., 2015). FM is a selective serotonin reuptake blocker and therefore may have resulted in an increase in 5-HT concentration in the synapse. This may have led to a decrease in the presynaptic release of 5HT in our stressed rats. Also, it is thought that in a 6-OHDA-lesioned rat, 5-HT receptors act similarly to antagonist receptors to reverse the effects of the neurotoxin (Huot et al., 2015). The early maternal separation experience and the 6-OHDA lesion may have disturbed monoamines and their transporters in the PFC to an extent that neuronal degeneration resulted in 5-HT, 5-HT transporters, and 5-HT receptors down-regulation. Taken together, our 5-HT analysis in the PFC suggests that many other factors such as the period (stress hyporesponsive period), the duration (chronic) and the type (maternal separation) of stress played a role in the turnover of 5-HT in maintaining normal brain function in the PFC. 5. Conclusion These data suggest a close functional relationship between the release of DA from terminals within the PFC and the retrieval of the learning and memory in rats. Overall, early treatment of cognitive impairment-induced exacerbation of Parkinsonism may be a promising strategy to delay a worsening of PD symptoms. These data suggest that DA and 5-HT signaling in the PFC are responsive to FM and may reduce stress-induced cognitive impairment in PD. Acknowledgements The work of the authors was supported by the National Research Foundation of South Africa, the University of KwaZulu-Natal and the College of Health Sciences. We would also like to thank the staff of the Biomedical Resource Centre of the University of KwaZulu-Natal for the technical assistance provided. References Aarsland, D., Kurz, M.W., 2010. The epidemiology of dementia associated with
80
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
Gironell, A., Pagonabarraga, J., Gich, I., 2008. Levodopa and executive performance in Parkinson’s disease: a randomized study. J. Int. Neuropsychol. Soc. 14, 832–841. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates. Academic Press. Pienaar, I.S., Kellaway, L.A., Russell, V.A., Smith, A.D., Stein, D.J., Zigmond, M.J., Daniels, W.M., 2008. Maternal separation exaggerates the toxic effects of 6hydroxydopamine in rats: implications for neurodegenerative disorders. Stress 11, 448–456. Sahin, E., Gumuslu, S., 2004. Alterations in brain antioxidant status: protein oxidation and lipid peroxidation in response to different stress models. Behav. Brain Res. 155, 241–248. Scatton, B., Javoy-Agid, F., Rouquier, L., Dubois, B., Agid, Y., 1983. Reduction of cortical dopamine, noradrenaline, serotonin and their metabolites in Parkinson’s disease. Brain Res. 275, 321–328. Scatton, B., Rouquier, L., Javoy-Agid, F., Agid, Y., 1982. Dopamine deficiency in the cerebral cortex in Parkinson disease. Neurology 32, 1039–1040. Solari, N., Bonito-Oliva, A., Fisone, G., Brambilla, R., 2013. Understanding cognitive deficits in Parkinson’s disease: lessons from preclinical animal models. Learn. Mem. 20, 592–600. Song, L., Che, W., Min-Wei, W., Murakami, Y., Matsumoto, K., 2006. Impairment of the spatial learning and memory induced by learned helplessness and chronic mild stress. Pharmacol. Biochem. Behav. 83, 186–193. Sun, Y.N., Wang, T., Wang, Y., Han, L.N., Li, L.B., Zhang, Y.M., Liu, J., 2015. Activation of 5-HT(1)A receptors in the medial subdivision of the central nucleus of the amygdala produces anxiolytic effects in a rat model of Parkinson’s disease. Neuropharmacology 95, 181–191. Takashima, A., Petersson, K.M., Rutters, F., Tendolkar, I., Jensen, O., Zwarts, M.J., Mcnaughton, B.L., Fernandez, G., 2006. Declarative memory consolidation in humans: a prospective functional magnetic resonance imaging study. Proc. Natl. Acad. Sci. U. S. A. 103, 756–761. Tao, C., Yan, W., Li, Y., Lu, X., 2016. Effect of antidepressants on spatial memory deficit induced by dizocilpine. Psychiatry Res. 244, 266–272. Tata, D.A., Markostamou, I., Ioannidis, A., Gkioka, M., Simeonidou, C., Anogianakis, G., Spandou, E., 2015. Effects of maternal separation on behavior and brain damage in adult rats exposed to neonatal hypoxia-ischemia. Behav. Brain Res. 280, 51–61. Tillerson, J.L., Cohen, A.D., Philhower, J., Miller, G.W., Zigmond, M.J., Schallert, T., 2001. Forced limb-use effects on the behavioral and neurochemical effects of 6hydroxydopamine. J. Neurosci. 21, 4427–4435. Vorhees, C.V., Williams, M.T., 2006. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat. Protoc. 1, 848–858. Williams, S.M., Goldman-Rakic, P.S., 1998. Widespread origin of the primate mesofrontal dopamine system. Cereb. Cortex 8, 321–345. Yamauchi, M., Miyara, T., Matsushima, T., Imanishi, T., 2006. Desensitization of 5-HT2A receptor function by chronic administration of selective serotonin reuptake inhibitors. Brain Res. 1067, 164–169. Yau, J.L., Noble, J., Hibberd, C., Rowe, W.B., Meaney, M.J., Morris, R.G., Seckl, J.R., 2002. Chronic treatment with the antidepressant amitriptyline prevents impairments in water maze learning in aging rats. J. Neurosci. 22, 1436–1442. Yuen, E.Y., Wei, J., Liu, W., Zhong, P., Li, X., Yan, Z., 2012. Repeated stress causes cognitive impairment by suppressing glutamate receptor expression and function of the prefrontal cortex. Neuron 73, 962–977.
Parkinson disease? Neurology 30, 1326–1330. Kulisevsky, J., Garcia-Sanchez, C., Berthier, M.L., Barbanoj, M., Pascual-Sedano, B., Gironell, A., Estevez-Gonzalez, A., 2000. Chronic effects of dopaminergic replacement on cognitive function in Parkinson’s disease: a two-year follow-up study of previously untreated patients. Mov. Disord. 15, 613–626. Liu, J., Wang, X., Mori, A., 1994. Immobilization stress-induced antioxidant defense changes in rat plasma: effect of treatment with reduced glutathione. Int. J. Biochem. 26, 511–517. Luo, X.M., Yuan, S.N., Guan, X.T., Xie, X., Shao, F., Wang, W.W., 2014. Juvenile stress affects anxiety-like behavior and limbic monoamines in adult rats. Physiol. Behav. 135, 7–16. Lupien, S.J., Mcewen, B.S., Gunnar, M.R., Heim, C., 2009. Effects of stress throughout the lifespan on the brain: behaviour and cognition. Nat. Rev. Neurosci. 10, 434–445. Mabandla, M.V., Nyoka, M., Daniels, W.M., 2015. Early use of oleanolic acid provides protection against 6-hydroxydopamine induced dopamine neurodegeneration. Brain Res. 1622, 64–71. Mabandla, M.V., Russell, V.A., 2010. Voluntary exercise reduces the neurotoxic effects of 6-hydroxydopamine in maternally separated rats. Behav. Brain Res. 211, 16–22. Madrigal, J.L., Olivenza, R., Moro, M.A., Lizasoain, I., Lorenzo, P., Rodrigo, J., Leza, J.C., 2001. Glutathione depletion: lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain. Neuropsychopharmacology 24, 420–429. Maeda, T., Kannari, K., Shen, H., Arai, A., Tomiyama, M., Matsunaga, M., Suda, T., 2003. Rapid induction of serotonergic hyperinnervation in the adult rat striatum with extensive dopaminergic denervation. Neurosci. Lett. 343, 17–20. Matsumoto, K., Yobimoto, K., Huong, N.T., Abdel-Fattah, M., Van Hien, T., Watanabe, H., 1999. Psychological stress-induced enhancement of brain lipid peroxidation via nitric oxide systems and its modulation by anxiolytic and anxiogenic drugs in mice. Brain Res. 839, 74–84. Morris, R., 1984. Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47–60. Mpofana, T., Daniels, W.M.U., Mabandla, M.V., 2014. Neuroprotective effects of caffeine on a maternally separated Parkinsonian rat model. J. Behav. Brain Sci. 04 (2), 8. Muck-Seler, D., Pivac, N., Diksic, M., 2012. Acute treatment with fluvoxamine elevates rat brain serotonin synthesis in some terminal regions: an autoradiographic study. Nucl. Med. Biol. 39, 1053–1057. Muller, T., Benz, S., Bornke, C., 2001. Delay of simple reaction time after levodopa intake. Clin. Neurophysiol. 112, 2133–2137. Narayanan, N.S., Land, B.B., Solder, J.E., Deisseroth, K., Dileone, R.J., 2012. Prefrontal D1 dopamine signaling is required for temporal control. Proc. Natl. Acad. Sci. U. S. A. 109, 20726–20731. Narayanan, N.S., Rodnitzky, R.L., Uc, E.Y., 2013. Prefrontal dopamine signaling and cognitive symptoms of Parkinson’s disease. Rev. Neurosci. 24, 267–268. Nikiforuk, A., Popik, P., 2011. Long-lasting cognitive deficit induced by stress is alleviated by acute administration of antidepressants. Psychoneuroendocrinology 36, 28–39. Nowakowska, E., Kus, K., Chodera, A., Rybakowski, J., 2001. Investigating potential anxiolytic: antidepressant and memory enhancing activity of deprenyl. J. Physiol. Pharmacol. 52, 863–873. Pani, L., Porcella, A., Gessa, G.L., 2000. The role of stress in the pathophysiology of the dopaminergic system. Mol. Psychiatry 5, 14–21. Pascual-Sedano, B., Kulisevsky, J., Barbanoj, M., Garcia-Sanchez, C., Campolongo, A.,
81
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Fluvoxamine maleate effects on dopamine signaling in the prefrontal cortex of stressed Parkinsonian rats: Implications for learning and memory Ernest Dalléa, Willie M.U. Danielsb, Musa V. Mabandlaa, a b
MARK
⁎
School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban 4000, South Africa School of Physiology, University of the Witwatersrand, Johannesburg, South Africa
A R T I C L E I N F O
A B S T R A C T
Keywords: Prefrontal cortex Cognitive impairment Dopamine Serotonin Parkinson’s disease
Parkinson’s disease (PD) is also associated with cognitive impairment and reduced extrinsic supply of dopamine (DA) to the prefrontal cortex (PFC). In the present study, we looked at whether exposure to early life stress reduces DA and serotonin (5-HT) concentration in the PFC thus leading to enhanced cognitive impairment in a Parkinsonian rat model. Maternal separation was the stressor used to develop an animal model for early life stress that has chronic effects on brain and behavior. Sprague-Dawley rats were treated with the antidepressant Fluvoxamine maleate (FM) prior to a unilateral 6-hydroxydopamine (6-OHDA) lesion to model motor deficits in rats. The Morris water maze (MWM) and the forelimb use asymmetry (cylinder) tests were used to assess learning and memory impairment and motor deficits respectively. Blood plasma was used to measure corticosterone concentration and prefrontal tissue was collected for lipid peroxidation, DA, and 5-HT analysis. Our results show that animals exposed to early life stress displayed learning and memory impairment as well as elevated basal plasma corticosterone concentration which were attenuated by treatment with FM. A 6-OHDA lesion effect was evidenced by impairment in the cylinder test as well as decreased DA and 5-HT concentration in the PFC. These effects were attenuated by FM treatment resulting in higher DA concentration in the PFC of treated animals than in non-treated animals. This study suggests that DA and 5-HT signaling in the PFC are responsive to FM and may reduce stress-induced cognitive impairment in PD.
1. Introduction Parkinson’s disease (PD) is a neurodegenerative condition in which dementia or cognitive dysfunction may precede the motor dysfunction that follows midbrain dopaminergic neuron loss (Narayanan et al., 2013). The prevalence of cognitive dysfunction is high with reports indicating this non-motor symptom to negatively affect the quality of life of about 60% of patients with PD and to cause morbidity and mortality in 36% of patients at the early onset of the disease (Cools et al., 2002; Foltynie et al., 2004; Forsaa et al., 2010; Solari et al., 2013). Patients suffering from PD, therefore, appear to be particularly susceptible to develop cognitive impairments with rates of 4–6 times that of normal aging being recorded (Aarsland et al., 2005; Narayanan et al., 2013). More importantly, studies have shown that cognitive impairment may occur at an early stage of PD and this seems to correlate well with dopaminergic dysfunction in the prefrontal cortex (PFC) (Brück et al., 2005; Chudasama and Robbins, 2006). However, to date, the cellular mechanism underlying the development of cognitive impairment in PD remains largely unknown.
⁎
Corresponding author. E-mail address: [email protected] (M.V. Mabandla).
http://dx.doi.org/10.1016/j.brainresbull.2017.05.014 Received 21 February 2017; Received in revised form 9 May 2017; Accepted 22 May 2017 Available online 24 May 2017 0361-9230/ © 2017 Published by Elsevier Inc.
The PFC is known to play a key role in short and long-term learning and memory processes (Takashima et al., 2006; Corcoran and Quirk, 2007). Deficits in cognitive planning and spatial working memory that occur in PD and progressively increase in intensity with time, have been associated with damage of the PFC (Cools et al., 2002; Solari et al., 2013). During the early stages (non-motor symptoms) of PD, cognitive impairment may correlate with dopaminergic dysfunction in the PFC, while during advanced stages (motor symptoms) of PD, the direct dopaminergic projection from the ventral tegmental area to the frontal cortex is affected resulting in cortical dopamine (DA) depletion (JavoyAgid and Agid, 1980; Scatton et al., 1983; Lupien et al., 2009; De la Fuente-Fernandez, 2012; Yuen et al., 2012; Solari et al., 2013). The PFC area of the brain receives a broad range of sensory and limbic inputs critical for appropriate behavioral task execution. However, altered DA transmission within the PFC results in abnormal information processing, leading to learning and memory deficits (Solari et al., 2013). In this regard, it has been suggested that learning and memory impairment may predict the development of dementia in PD that is so highly prevalent across the entire course of the disease (Aarsland et al., 2005;
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
2.2. Drugs and reagents
Aarsland and Kurz, 2010). We have recently shown that the antidepressant Fluvoxamine maleate (FM) is not only useful in treating major anxiety/depressivelike symptoms but also attenuates neurotoxin induced-DA degeneration in an animal model for PD (Dallé et al., 2017). The effects of antidepressants on serotonergic and dopaminergic transmission are well established (Tao et al., 2016). These effects include increasing serotonin (5-HT) and DA levels in the striatum and the PFC (Lupien et al., 2009; Hemmerle et al., 2012). More importantly, the effects of antidepressants on cognition are widely known since long-lasting cognitive deficits induced by stress can be attenuated by some antidepressant (Nikiforuk and Popik, 2011). However, finding the mechanism of cognitive dysfunction in PD is critical for treating cognitive symptoms of PD, since to date, only few effective treatments are available. Moreover, to the best of our knowledge, apart from Levodopa that has been shown to improve cognitive symptoms of PD depending on the stage of the disease and the integrity of striatal DA signaling, no studies have looked at the effects of an antidepressant to treat PD-induced DA depletion in the PFC (Kulisevsky et al., 2000; Cools et al., 2002; Muller et al., 2001; Cools, 2006; Pascual-Sedano et al., 2008). We hypothesized that chronic treatment with FM will reduce learning and memory deficits and counteracts 6-OHDA-induced Parkinsonian symptoms in stressed rats. The present study consequently aimed to investigate the effects of FM treatment on cognitive deficits in a Parkinsonian rat model and whether serotonergic and/or dopaminergic mechanisms of FM in the PFC could contribute to an antiparkinsonian effect in stressed rats.
The drug Fluvoxamine maleate, Temgesic and Biotane were obtained from Pharmed Pharmaceuticals LTD (Rochdale Park, Durban, South Africa). Desipramine (D3900), atropine, pentobarbital and 6OHDA were purchased from Sigma (St. Louis MO, USA). The Corticosterone (RE52211), Dopamine (RE59161) and Serotonin (RE59121) ELISA kits were purchased from IBL International GmbH (Hamburg, Germany). The lipid peroxidation (MDA) assay kit (K739100) was obtained from BioVision (Mountain view, CA, USA). 2.3. Maternal separation Maternal separation was the stressor used to enhance learning and memory deficits (non-motor symptoms of PD) as per study by Lupien et al. (2009) and Hermmerle et al. (2012). This protocol was also used so as to investigate whether addressing these symptoms at their early stage with FM could delay the motor symptoms in a model of PD. The maternal separation stress protocol was carried out once a day from PND 2 to PND 14. We used a stress protocol previously described in Mabandla and Russell (2010) and Dalle et al. (2016). Briefly, the pups were taken away from their dams and kept in a separate room for 3 h (09:00–12:00). All normally reared pups were left undisturbed with their dams. 2.4. Behavioral tests Behavioral tests were used to assess the effects of FM on learning and memory in a maternally separated rat as well as to assess the effects of the drug on motor dysfunction in a Parkinsonian rat model. The behavioral tests used included the MWM test and the limb-use asymmetry test (cylinder test). The tests were performed pre- as well as post-lesion with 6-OHDA. All behavioral tests were video-recorded for subsequent scoring and manually analyzed by an evaluator blind to the study.
2. Materials and methods 2.1. Animals The experimental protocol used in this study was reviewed and approved by the Animal Research Ethics Committee of the University of KwaZulu-Natal (018/15/Animal) in accordance with the guidelines of the National Institutes of Health, USA. The sample size was set according to previous studies where the statistical power was shown (Eng, 2003; Dalle et al., 2016). A total of 60 male Sprague-Dawley rats obtained from the Biomedical Resource Unit of the University of KwaZulu-Natal were used in this study. They were housed in polypropylene cages (38 × 32 × 16 cm) under controlled temperature (21 ± 2 °C) and humidity (55–60%). Food and water were freely available. The daily light/dark cycle was 07:00–19:00 (Mabandla and Russell, 2010). On post-natal day (PND) 1, the rats were sexed and culled to 6 male pups per litter and randomly divided into 6 equal groups as follows: non-stressed (NS), non-stressed treated with saline (NSS), non-stressed treated with FM (NSF), maternally separated (MS), maternally separated treated with saline (MSS) and maternally separated treated with FM (MSF). The rats were weaned on PND 21 after which they were kept 6 per cage (Dalle et al., 2016). On PND 29, all treated groups received saline or FM intraperitoneally from PND 29–59 once daily. NSS and MSS rats received vehicle injections (saline 1 ml/ 250 g of body weight) and were lesioned with saline to control for the effects of handling, stress, lesion and the injection itself. NSS and MSS data were not included since no significant effect was found between saline treated rats and non-treated rats. Lesion refers to the intracerebral injection of the neurotoxin 6-OHDA on PND 60 in all groups. The animals were weighed prior to all experimental procedures and were brought to the experimental room at least 1 h before experimentation. The learning and memory ability test was assessed on PND 28, 58 and 74 using the Morris water maze (MWM) apparatus (Vorhees and Williams, 2006; Beraki et al., 2009). Limb-use asymmetry was assessed on PND 59 and 75 using the cylinder test (Mabandla and Russell, 2010). The rats were sacrificed on PND 76. All experimental procedures were conducted between 09:00 and 16:00.
2.4.1. Morris water maze (MWM) test The MWM test was used to assess spatial learning and memory (D’hooge and De Deyn, 2001). The MWM apparatus used was a circular tank (1 m in diameter) that is divided into 4 quadrants filled with water (22–23° C) in where a hidden square plexiglass platform (10 × 10 cm wide and 20 cm high) is submerged (1 mm) bellow the water surface. The hidden platform was placed in one of the quadrants and each quadrant had a visual cue to help the rat in its navigation. All animals were exposed to training sessions (4 trials per day) over consecutive days (PND 25–27) (Vorhees and Williams, 2006). The latency time (time taken to reach the hidden platform) was recorded and was taken as the learning process (Morris, 1984). A probe test was conducted (without the platform) to assess the ability of the rats to remember the quadrant in which the platform was located (Garthe and Kempermann, 2013). 2.4.2. The limb-use asymmetry test (Cylinder test) The cylinder test was used to assess the forelimb used during exploratory activity in the plexiglass cylinder and for landing over a period of 5 min. As the neurotoxin 6-OHDA was injected into the left hemisphere, a successful model of Parkinsonism would result in a bias towards left forelimb use after lesion (Tillerson et al., 2001; Mabandla and Russell, 2010). The cylinder was made of transparent plexiglass (20 cm in diameter and 30 cm in height). The rats were assessed for percentage limb-use of the impaired (right) limb by using the following equation: % limb use of impaired = [(impaired + ½ both)/(impaired + unimpaired + both)] × 100. Both, refers to the use of both the impaired and unimpaired limbs during exploratory activity (Tillerson et al., 2001). 76
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
300 μl of the Malondialdehyde (MDA) lysis buffer was homogenized on ice using a sonicator (CML-4, Fisher, USA) before being centrifuged at 13000 rpm for 10 min. The supernatant (200 μl) from each homogenized sample was pipetted into a microcentrifuge tube. To prepare the standards, 10 μl of MDA standard was diluted with 407 μl of bidistilled water so as to prepare a solution of 0.1 M of MDA. 0.1 M MDA (20 μl) solution was diluted with bidistilled water (980 μl) resulting in a 2 mM MDA standard. Thiobarbituric acid (TBA) solution (600 μl) was then added to the vials containing the Standards and samples. This was followed by incubation for 1 h in a water bath at 95° C. Vials containing the Standards as well as the control samples were allowed to cool at room temperature for 10 min after which 200 μl of each solution (Standards and samples) was pipetted in duplicate into a 96-well microplate. Absorbance was read at 532 nm using a spectrophotometer (Spectrostar Nano BMG LABTECH, Germany).
2.5. Fluvoxamine maleate (FM) treatment Prior to treatment, the rats were weighed and the volume of FM to be injected (1 ml/250 g of body weight) was calculated accordingly. FM was always freshly prepared by dissolving in saline after which it was intraperitoneally (i.p) injected (25 mg/kg) once daily during the treatment period (Gerstenberg et al., 2003; Muck-Seler et al., 2012; Dalle et al., 2016). 2.6. Hemiparkinsonian rat model (6-OHDA lesion) 6-OHDA was injected stereotaxically into the medial forebrain bundle (MFB) as per Paxinos and Watson, (1998). The norepinephrine reuptake blocker desipramine HCl (15 mg/kg, i.p.) was injected 30 min prior to 6-OHDA infusion in order to inhibit 6-OHDA uptake by noradrenergic neurons (Goodman et al., 1990). Before 6-OHDA infusion, the rats were anaesthetized with sodium pentobarbital (60 mg/kg, i.p.) followed by an atropine (0.2 mg/kg, i.p.) injection so as to facilitate respiration while the rat was unconscious. This was followed by shaving the head before positioning it in the stereotaxic apparatus (David Kopf Instruments, Tujunga CA, USA). The skin covering the scalp was disinfected with Biotane and a midline incision was made to expose the skull. A burr hole was drilled on the skull at the following coordinates: anterior-posterior (AP) = +4.7 mm anterior to lambda; medio-lateral (ML) = +1.6 mm from midline suture (Paxinos and Watson, 1998). At these coordinates, a Hamilton syringe was lowered 8.4 mm ventral to dura before 6-OHDA HCl (5 μg/4 μl) dissolved in 0.2% ascorbic acid was injected into the left MFB over a period of 8 min. The needle was kept in the MFB for 1 min prior to the injection and for 2 min following the injection so as to maximize diffusion of the neurotoxin. After the needle was retracted, the incision was sutured and cleaned and the rat was kept warm under a heating lamp so as to prevent hypothermia during recovery. The rats were injected with the analgesic Temgesic (0.05 mg/kg subcutaneously) before being returned to their home cages.
2.7.2.2. Dopamine (DA) in the PFC. Dissected PFC tissue was weighed and placed in Eppendorf tubes prior to freezing in liquid nitrogen before being stored at −80 °C in a bio-freezer until the day of analysis. On the day of analysis, the PFC tissue (n = 6/group) was removed from the bio-freezer and allowed to thaw at room temperature. EDTA–HCL buffer (500 μl) was added to each tube containing PFC tissue. The tissue was homogenized in a sonicator before being centrifuged at 3500 rpm for 10 min at 4 °C. The supernatant was pipetted into new Eppendorf tubes for DA analysis using a dopamine ELISA kit according to the manufacturer’s protocol. The DA analysis protocol consisted of an extraction procedure that was followed by quantification. Both steps were conducted on the same day. Briefly, extraction consisted of pipetting 20 μl of each standard, 20 μl of each control and 500 μl of each sample into respective wells in the extraction plate. The standards and controls were diluted with bidistilled water in order to correct for differences in volume as per the manufacturer’s instructions. For the quantification procedure, the extracted standards and controls were diluted with 500 μl of release buffer. Pre-diluted standards, controls and 100 μl of each extracted sample (without pre-dilution) were pipetted into respective wells containing COMT Enzyme solution (75 μl) supplied with the kit. All samples, standards and controls were analysed in duplicate. The optical density of the samples was measured using a microtiter plate reader (SPECTROstar Nano, BMG LABTECH GmbH, Ortenberg, Germany) at 405 nm, within 10 min adding the stop solution.
2.7. Decapitation and neurochemistry On PND 76 (16 days post-lesion with 6-OHDA), the rats were decapitated using a sharp guillotine. Trunk blood and PFC tissue were collected. Trunk blood was collected in EDTA coated test tubes and immediately centrifuged using a refrigerated centrifuge (Labofurge 200, Heraeus Sepatech, Hanau, Germany). Plasma was transferred into Eppendorf tubes and snap frozen in liquid nitrogen before being stored in a bio-freezer at −80 °C until the day of analysis. Plasma corticosterone concentration (n = 10/group) was analyzed using a corticosterone ELISA kit according to the manufacturer’s protocol.
2.7.2.3. Serotonin (5-HT) in the PFC. Hydrochloric acid (0.05 M) mixed with 0.1% ascorbic acid was added to each sample (n = 6/group), homogenized with a sonicator then shaken (protected from sun light) for 1 h before being centrifuged for 20 min at 4000 rpm. The supernatant was collected into new Eppendorf tubes. Standards, controls, and samples (25 μl each) were pipetted into reaction tubes provided with the kit. Acylation Buffer (500 μl) and Acylation Reagent (25 μl) were added to all tubes, mixed and incubated for 15 min at room temperature. The acylated standards, controls, and samples (25 μl each) were pipetted into respective wells of the 5-HT microtiter strips (provided with the kit). 5-HT antiserum (100 μl) was pipetted into each well, incubated for 30 min after which each well was washed with 300 μl wash buffer and then dried. Thereafter, 100 μl of Conjugate solution was pipetted into each well, allowed to incubate for 15 min. The contents were then discarded after which the well was washed and dried. A volume of 100 μl of Substrate solution was pipetted into each well and allowed to incubate for 15 min before the Stop solution (100 μl) was added. All samples, standards, and controls were read in duplicate. The absorbance of the solution in the wells was read within 10 min using a microplate reader set at 450 nm.
2.7.1. Plasma corticosterone concentration On the day of analysis, blood plasma samples were allowed to thaw on ice. A volume of 20 μl of each standard, control, and plasma samples was dispensed into the appropriate wells of a 96 well plate. Enzyme conjugate (200 μl) was added to each well, mixed and incubated for 1 h at room temperature. Following the addition of Substrate solution (100 μl) to each well, the plate was incubated at room temperature for 15 min. Following incubation, Stop solution (50 μl) was added after which the optical density of the standards, controls, and samples was measured using a microtiter plate reader (SPECTROstar Nano, BMG LABTECH GmbH, Ortenberg, Germany) at 450 nm within 10 min as per the manufacturer’s protocol. 2.7.2. Brain tissue 2.7.2.1. Lipid peroxidation. Lipid peroxidation (Malondialdehyde) quantification as a measure of oxidative stress was evaluated in the PFC of all groups (n = 6/group) using the method described in Hall and Bosken (2009) and the manufacturer’s protocol. Briefly, PFC tissue in 77
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
2.8. Statistical analysis All results are presented as mean ± SEM. The data was analyzed using the software program GraphPad Prism (version 5.0, San Diego, California, USA). Data normality was assessed by the KolmogorovSmirnov test and where data met requirements, parametric or nonparametric tests were used. The main factors in each analysis included stress and treatment. When measuring only one variable (stress versus non-stressed), a paired t-test was used for analysis. Two-way analysis of variance (ANOVA) was used for behavior as well as corticosterone, lipid peroxidation, DA and 5-HT concentration data analysis. In each analysis, significant main effects were followed by Bonferroni post hoc test. p < 0.05 was considered significant in all analysis. 3. Results Fig. 1. Time taken by maternally separated and non-stressed rats to find the hidden platform in the MWM on the training days (PND 25, 26 and 27) and on the test day (PND 28). **(Day 1 vs. Day 2, p = 0.0038), ***(Day 2 vs. Day 3, p = 0.0001), and ***(Day 1 vs. Day 3, p < 0.0001), Paired t-tests in non-stressed rats. *(Day 1 vs. Day 2, p = 0.0149) and ***(Day 1 vs. Day 3, p = 0.0001). Paired t-tests in maternally separated rats. *(NS Day 3 vs. MS Day 3, p = 0.0107, Paired t tests). Data are presented as mean ± SEM. (n = 20/group). On PND 28: ***(NS vs. MS, p = 0.0001, paired t-test). Data are presented as mean ± SEM. (n = 20/group). Effects of FM in the memory ability of maternally separated and non-stressed rats with regards to the time spent in the quadrant of the hidden platform in the MWM on PND 59. *(NS vs. MS, p = 0.0107, paired t-test), [*(NS vs. NSF), F (1, 36) = 8.963, p = 0.050 and **(MS vs. MSF), F(1, 36) = 11.16, p = 0.0020, two-way ANOVA with Bonferroni post hoc test]. Data are presented as mean ± SEM. (n = 10/group). Effects of FM in the memory ability of maternally separated and non-stressed rats with regards to the time spent in the quadrant of the hidden platform in the MWM on PND 74. ***(NS vs. MS, p < 0.0001, paired ttest), and [***(MS vs. MSF), F(1, 36) = 14.88, p = 0.0005, two-way ANOVA with Bonferroni post hoc test]. Data are presented as mean ± SEM. (n = 10/group).
3.1. Morris water maze (MWM) test Training sessions on the MWM took place on PND 25–27. A training effect was present in the non-stressed groups **(Day 1 vs. Day 2 p = 0.0038), ***(Day 2 vs. Day 3, p = 0.0001) and ***(Day 1 vs. Day 3, p < 0.0001). A similar effect was present in the stressed groups *(Day 1 vs. Day 2, p = 0.0149) and ***(Day 1 vs. Day 3, p = 0.0001). Overall, non-stressed rats had a shorter latency on day 3 *(NS Day 3 vs. MS Day 3, p = 0.0107). The effects of maternal separation on short and long-term learning and memory were assessed on PND 28, 58 and 74. On PND 28 (pretreatment stage), there was a stress effect as maternally separated rats took longer to find the platform ***(NS vs. MS, p = 0.0001). On PND 58, the probe test showed that there was a stress effect as maternally separated rats struggled to find the quadrant with the hidden platform *(NS vs. MS, p = 0.0107). However, there was a treatment effect as FM-treated rats spent more time in the quadrant with the hidden platform in both non-stressed and maternally separated rats [*(NS vs. NSF), F (1, 36) = 8.963, p = 0.050 and **(MS vs. MSF), F(1, 36) = 11.16, p = 0.0020]. Similarly, on PND 74, there was a stress effect as maternally separated rats spent less time in the quadrant of the hidden platform when compared to non-stressed rats *(NS vs. MS, p = 0.0171). A treatment effect on memory was observed as FM-treated rats spent more time in the quadrant of the hidden platform in maternally separated rats [*(MS vs. MSF), F(1, 36) = 3.868, p = 0.0333]. Data shown in Fig. 1. 3.2. Cylinder test Forelimb use asymmetry was assessed on PND 58 and PND 75. Prior to 6-OHDA lesion (PND 58), no asymmetry was observed in all the rats (Data not shown). On PND 75, a stress effect was evident as 6-OHDA lesion in these rats resulted in decreased use of the right (impaired) forelimb when touching the wall of the cylinder *(NS vs MS, p = 0.0199). A treatment effect was present as FM treatment reduced forelimb use asymmetry caused by the 6-OHDA lesion in both the nonstressed and maternally separated rats [*(NS vs. NSF, F(1, 36) = 6.196, p = 0.0176) and **(MS vs. MSF), F(1, 36) = 16.65, p = 0.0002]. In addition, significant main effects of stress and FM treatment with respect to the use of the right (impaired) forelimb for landing was observed *(NS vs MS, p = 0.0269), [**(NS vs. NSF), F(1, 36) = 12.59, p = 0.0011] and **(MS vs. MSF), F(3, 36) = 7.708, p = 0.0022]. Data presented in Fig. 2.
Fig. 2. Number of times the rat used the impaired (right) forelimb to touch the wall of the cylinder and to land on the floor expressed as a percentage of the total number of times it touched the wall of the cylinder or landed on the floor of the cylinder on PND 75. All groups were lesioned with 6-OHDA on PND 60. Wall touch, *(NS vs MS, p = 0.0199, paired t-test), [*(NS vs. NSF, F(1, 36) = 6.196, p = 0.0176) and **(MS vs. MSF), F(1, 36) = 16.65, p = 0.0002, two-way ANOVA with Bonferroni post hoc test]. Landing on the floor, *(NS vs MS, p = 0.0269, paired t-test), [**(NS vs. NSF), F(1, 36) = 12.59, p = 0.0011] and ***(MS vs. MSF), F(3, 36) = 7.708, p = 0.0022, two-way ANOVA with Bonferroni post hoc test]. Values are expressed as mean ± SEM (n = 10/group).
elevated than those of the non-stressed rats ***(NS vs. MS, p = 0.0002). A treatment effect was found as FM treatment significantly decreased plasma corticosterone levels in maternally separated rats [**(MS vs. MSF), F(1, 36) = 9.84, p = 0.0039]. In addition, as one of the principal mechanism of neuronal injury associated with oxygen radicals in PD, lipid peroxidation levels were measured to quantify oxidative stress and to evaluate the antioxidant capacity of FM. A stress effect was observed ***(NS vs. MS, p = 0.0007). Treatment with FM
3.3. Corticosterone and lipid peroxidation concentration Plasma corticosterone and Lipid peroxidation concentration were measured on PND 76. We found a stress effect as the plasma corticosterone levels of maternally separated rats was significantly 78
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
enhanced cognitive impairment in a Parkinsonian rat model. The MWM test showed that exposure to early life stress resulted in learning and memory deficits. We also found that exposure to early life stress exacerbated the effects of 6-OHDA on motor function (Mpofana et al., 2014; Mabandla and Russell, 2010; Dalle et al., 2016). These results are in agreement with previous studies and provide evidence that exposure to maternal separation impairs learning and memory (Solari et al., 2013; Aisa et al., 2007; Pienaar et al., 2008; Tata et al., 2015). However, treatment with the antidepressant FM improved learning and memory impairment in both the non-stressed and stressed groups. Although this finding is the first to find FM’s therapeutic effect in relieving cognitive impairment in a rat model of PD, there is evidence suggesting that impaired learning and memory could be the result of elevated glucocorticoid (corticosterone) levels (Fann et al., 2001; Nowakowska et al., 2001; Harmer et al., 2002; Yau et al., 2002). Studies have shown that early maternal separation disturbs the hypothalamic-pituitary-adrenal (HPA) axis and leaves the brain more vulnerable to subsequent traumatic events later in life (Lupien et al., 2009; Mpofana et al., 2014). Reducing the cumulative lifetime exposure to corticosterone excess by long-term exposure to FM may have prevented the emergence of cognitive deficits in our rats. We, therefore, speculate that deficits may have occurred as a result of malfunctioning corticosterone regulation which is known to have a critical role in the development of cognitive function (Duman et al., 2000; Song et al., 2006). In the cylinder test, as expected, our results showed a preference to using the unimpaired forelimb in both groups (NS and MS) following 6OHDA lesion. However, exposure to early life stress exacerbated this asymmetry. These results are in agreement with previous findings that used the same animal model (Mabandla and Russell, 2010; Mpofana et al., 2014; Dalle et al., 2016). We further found that treatment with FM reduced this asymmetrical use of the forelimbs suggesting that FM treatment may be neuroprotective against DA degeneration in the nigrostriatal pathway. We have recently shown that FM possesses antiinflammatory and antioxidant properties (Dallé et al., 2017). Although further specific investigations need to be done, these properties may have been essential in ameliorating the toxic effects of 6-OHDA in NSF and MSF rats through reinforcement of enzymatic anti-inflammatory and antioxidant defenses (Mabandla et al., 2015). Plasma corticosterone concentration was measured to evaluate the effectiveness of our early life model for stress. Our results did indeed show that we have a stressed animal model with basal corticosterone concentration being higher in the maternally separated rats (Daniels et al., 2004). However, there was a reduction in plasma corticosterone concentration in the stressed rats following treatment with FM. It has been shown that FM can activate the HPA axis resulting in neuroendocrine responses via specific serotonergic neurons (Yamauchi et al., 2006). As learning and memory deficits as well as increased corticosterone concentration have been linked to anxiety-like behavior, FM’s ability to decrease corticosterone concentration in maternally separated rats confirms the anxiolytic effect of the drug as shown by Hiemke and Hartter (2000) and Irons (2005). Studies have shown that exposure to stress induces lipid peroxidation in the brain (Liu et al., 1994; Matsumoto et al., 1999; Madrigal et al., 2001). The positive correlation between plasma corticosterone concentration and lipid peroxidation suggests that elevated corticosterone concentration plays a role in increasing lipid peroxidation (Sahin and Gumuslu, 2004). We found that exposure to stress resulted in increased lipid peroxidation. However, FM treatment resulted in decreased lipid peroxidation levels in the PFC of stressed rats. These results are in agreement with results from previous studies that showed that antidepressants decreased oxidative stress in the PFC of stressed animals (Cline et al., 2015). We, therefore, suggest that the antioxidant effects of FM may be responsible for mopping up the free radicals generated by the autooxidation of 6-OHDA. In PD, reduction of cortical dopaminergic transmission can be
Fig. 3. Effect of FM on plasma corticosterone concentration in 6-OHDA lesioned, maternally separated (MS, MSF) and non-stressed (NS, NSF) rats on PND 76. ***(NS vs. MS, p = 0.0002, paired t-test) and [**(MS vs. MSF), F(1, 36) = 9.84, p = 0.0039, twoway ANOVA with Bonferroni post hoc test]. Data presented as mean ± SEM. (n = 10/ group). Effect of FM on lipid peroxidation levels in 6-OHDA lesioned, maternally separated (MS, MSF) and non-stressed (NS, NSF) rats on PND 76. ***(NS vs. MS, p = 0.0007, paired t-test) and [**(MS vs. MSF), F(1, 20) = 14.43, p = 0.0011, two-way ANOVA with Bonferroni post hoc test]. Data presented as mean ± SEM. (n = 6/group).
significantly decreased lipid peroxidation levels in maternally separated rats [**(MS vs. MSF), F(1, 20) = 14.43, p = 0.0011]. Data presented in Fig. 3. 3.4. Dopamine (DA) and serotonin (5-HT) concentration DA and 5-HT concentration were measured on PND 76. A stress effect that resulted in decreased DA concentration in the maternally separated rats was observed *(NS vs. MS, p = 0.0385). Treatment with FM attenuated the decrease in DA concentration in maternally separated rats [*(MS vs. MSF), F(1, 20) = 6.627, p = 0.0181]. In addition, a stress effect resulting in decreased 5-HT concentration was observed *(NS vs. MS, p = 0.0219). Treatment with FM affected 5-HT transmission in non-stressed rats [**(NS vs. NSF), F(1, 20) = 11.21, p = 0.0032]. Data presented in Fig. 4. 4. Discussion In the present study, we looked at whether exposure to early life stress exacerbates DA and 5-HT depletion in the PFC thus leading to
Fig. 4. Effect of FM on DA concentration in 6-OHDA lesioned, maternally separated (MS, MSF) and non-stressed (NS, NSF) rats on PND 76. *(NS vs. MS, p = 0.0385, paired t-test) and [*(MS vs. MSF), F(1, 20) = 6.627, p = 0.0181, two-way ANOVA with Bonferroni post hoc test]. Data presented as mean ± SEM. (n = 6/group). Effect of FM on 5-HT concentration in 6-OHDA lesioned, maternally separated (MS, MSF) and non-stressed (NS, NSF) rats on PND 76. *(NS vs. MS, p = 0.0219, paired t-test) and [**(NS vs. NSF), F(1, 20) = 11.21, p = 0.0032, two-way ANOVA with Bonferroni post hoc test]. Data presented as mean ± SEM. (n = 6/group).
79
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
Parkinson’s disease. Brain Pathol. 20, 633–639. Aarsland, D., Zaccai, J., Brayne, C., 2005. A systematic review of prevalence studies of dementia in Parkinson’s disease. Mov. Disord. 20, 1255–1263. Aisa, B., Tordera, R., Lasheras, B., Del Rio, J., Ramirez, M.J., 2007. Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats. Psychoneuroendocrinology 32, 256–266. Arnsten, A.F., Raskind, M.A., Taylor, F.B., Connor, D.F., 2015. The effects of stress exposure on prefrontal cortex: translating basic research into successful treatments for post-traumatic stress disorder. Neurobiol. Stress 1, 89–99. Belujon, P., Grace, A.A., 2015. Regulation of dopamine system responsivity and its adaptive and pathological response to stress. Proc. R. Soc. B: Biol. Sci. 282. Beraki, S., Diaz-Heijtz, R., Tai, F., Ogren, S.O., 2009. Effects of repeated treatment of phencyclidine on cognition and gene expression in C57BL/6 mice. Int. J. Neuropsychopharmacol. 12, 243–255. Brück, A., Aalto, S., Nurmi, E., Bergman, J., Rinne, J.O., 2005. Cortical 6- [18F]fluoro-ldopa uptake and frontal cognitive functions in early Parkinson’s disease. Neurobiol. Aging 26, 891–898. Buoli, M., Cumerlato Melter, C., Caldiroli, A., Altamura, A.C., 2015. Are antidepressants equally effective in the long-term treatment of major depressive disorder? Hum. Psychopharmacol. 30, 21–27. Chudasama, Y., Robbins, T.W., 2006. Functions of frontostriatal systems in cognition: comparative neuropsychopharmacological studies in rats monkeys and humans. Biol. Psychol. 73, 19–38. Cline, B.H., Anthony, D.C., Lysko, A., Dolgov, O., Anokhin, K., Schroeter, C., Malin, D., Kubatiev, A., Steinbusch, H.W., Lesch, K.P., Strekalova, T., 2015. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav. Brain Res. 276, 118–129. Cools, R., 2006. Dopaminergic modulation of cognitive function-implications for L-DOPA treatment in Parkinson’s disease. Neurosci. Biobehav. Rev. 30, 1–23. Cools, R., Stefanova, E., Barker, R.A., Robbins, T.W., Owen, A.M., 2002. Dopaminergic modulation of high-level cognition in Parkinson’s disease: the role of the prefrontal cortex revealed by PET. Brain 125, 584–594. Corcoran, K.A., Quirk, G.J., 2007. Activity in prelimbic cortex is necessary for the expression of learned, but not innate, fears. J. Neurosci. 27, 840–844. D’hooge, R., De Deyn, P.P., 2001. Applications of the Morris water maze in the study of learning and memory. Brain Res. Brain Res. Rev. 36, 60–90. Dalle, E., Daniels, W.M., Mabandla, M.V., 2016. Anti-parkinsonian effects of fluvoxamine maleate in maternally separated rats. Int. J. Dev. Neurosci. 53, 26–34. Dallé, E., Daniels, W.M.U., Mabandla, M.V., 2017. Fluvoxamine maleate normalizes striatal neuronal inflammatory cytokine activity in a parkinsonian rat model associated with depression. Behav. Brain Res. 316, 189–196. Daniels, W.M., Pietersen, C.Y., Carstens, M.E., Stein, D.J., 2004. Maternal separation in rats leads to anxiety-like behavior and a blunted ACTH response and altered neurotransmitter levels in response to a subsequent stressor. Metab. Brain Dis. 19, 3–14. De la Fuente-Fernandez, R., 2012. Frontostriatal cognitive staging in Parkinson’s disease. Parkinson’s Dis. 561046. Duman, R.S., Malberg, J., Nakagawa, S., D’Sa, C., 2000. Neuronal plasticity and survival in mood disorders. Biol. Psychiatry 48, 732–739. Dymecki, J., Lechowicz, W., Bertrand, E., Szpak, G.M., 1996. Changes in dopaminergic neurons of the mesocorticolimbic system in Parkinson’s disease. Folia Neuropathol. 34, 102–106. Eng, J., 2003. Sample size estimation: how many individuals should be studied? Radiology 227, 309–313. Fann, J.R., Uomoto, J.M., Katon, W.J., 2001. Cognitive improvement with treatment of depression following mild traumatic brain injury. Psychosomatics 42, 48–54. Foltynie, T., Brayne, C.E., Robbins, T.W., Barker, R.A., 2004. The cognitive ability of an incident cohort of Parkinson’s patients in the UK. The CamPaIGN study. Brain 127, 550–560. Forsaa, E.B., Larsen, J.P., Wentzel-Larsen, T., Alves, G., 2010. What predicts mortality in Parkinson disease?: a prospective population-based long-term study. Neurology 75, 1270–1276. Garthe, A., Kempermann, G., 2013. An old test for new neurons: refining the Morris water maze to study the functional relevance of adult hippocampal neurogenesis. Front. Neurosci. 7, 63. Gerstenberg, G., Aoshima, T., Fukasawa, T., Yoshida, K., Takahashi, H., Higuchi, H., Murata, Y., Shimoyama, R., Ohkubo, T., Shimizu, T., Otani, K., 2003. Relationship between clinical effects of fluvoxamine and the steadystate plasma concentrations of fluvoxamine and its major metabolite fluvoxamino acid in Japanese depressed patients. Psychopharmacology 167, 443–448. Goodman, W.K., Price, L.H., Delgado, P.L., Palumbo, J., Krystal, J.H., Nagy, L.M., Rasmussen, S.A., Heninger, G.R., Charney, D.S., 1990. Specificity of serotonin reuptake inhibitors in the treatment of obsessive-compulsive disorder. Comparison of fluvoxamine and desipramine. Arch. Gen. Psychiatry 47, 577–585. Hall, E.D., Bosken, J.M., 2009. Measurement of oxygen radicals and lipid peroxidation in neural tissues. Curr. Protoc. Neurosci. 1–51 Chapter 7, Unit 7.17. Harmer, C.J., Bhagwagar, Z., Cowen, P.J., Goodwin, G.M., 2002. Acute administration of citalopram facilitates memory consolidation in healthy volunteers. Psychopharmacology (Berl.) 163, 106–110. Hiemke, C., Hartter, S., 2000. Pharmacokinetics of selective serotonin reuptake inhibitors. Pharmacol. Ther. 85, 11–28. Huot, P., Fox, S.H., Brotchie, J.M., 2015. Monoamine reuptake inhibitors in Parkinson’s disease. Parkinsons Dis. 2015, 609428. Irons, J., 2005. Fluvoxamine in the treatment of anxiety disorders. Neuropsychiatr. Dis. Treat. 1, 289–299. Javoy-Agid, F., Agid, Y., 1980. Is the mesocortical dopaminergic system involved in
associated with cognitive deficits (Scatton et al., 1982, 1983; Kulisevsky et al., 2000). Our results show that DA concentration was lower in the PFC of the stressed rats. This suggests that DA concentration is responsive to stress and may modulate its effects via the mesocortical pathway in agreement with previous studies that showed that stress alters DA metabolism in the PFC (Pani et al., 2000; Luo et al., 2014; Arnsten et al., 2015). The PFC plays a role in controlling the HPA axis’ responds to stress by modulating the neuroendocrine function via the mesocortical pathway (projections from the ventral tegmental area to the PFC) (Narayanan et al., 2012; Belujon and Grace, 2015). In fact, the PFC region receives DA projections from the ventral tegmental and medial nigral regions of the midbrain which may also degenerate in the course of PD (Javoy-Agid and Agid, 1980; Dymecki et al., 1996; Williams and Goldman-Rakic, 1998). Our results suggest that maternal separation resulted in suppression of the limbic DA transmission in stressed rats. However, FM treatment preserved DA transmission in the PFC of stressed rats suggesting a neuroprotective effect of the drug. Cools et al. (2002) reported that increases in DA transmission in the PFC contribute to cognitive stability in predicting the consequences of caudate nucleus dysfunction. Therefore, it is possible that similar to Levodopa, FM may have induced blood flow increases in the PFC regions so as to preserve cortical dopaminergic transmission. We also found that the concentration of 5-HT was lower in the stressed animals. These results agree with previous findings that showed that 6-OHDA lesioned maternally separated rats have altered 5-HT concentration in the PFC (Daniels et al., 2004; Sun et al., 2015). However, FM treatment did not affect 5-HT concentration in stressed animals. We speculated that serotonergic hyperinnervation may have occurred in NSF rats but not in MSF, and this may have compensated for the dopaminergic denervation (Maeda et al., 2003; Buoli et al., 2015; Huot et al., 2015). FM is a selective serotonin reuptake blocker and therefore may have resulted in an increase in 5-HT concentration in the synapse. This may have led to a decrease in the presynaptic release of 5HT in our stressed rats. Also, it is thought that in a 6-OHDA-lesioned rat, 5-HT receptors act similarly to antagonist receptors to reverse the effects of the neurotoxin (Huot et al., 2015). The early maternal separation experience and the 6-OHDA lesion may have disturbed monoamines and their transporters in the PFC to an extent that neuronal degeneration resulted in 5-HT, 5-HT transporters, and 5-HT receptors down-regulation. Taken together, our 5-HT analysis in the PFC suggests that many other factors such as the period (stress hyporesponsive period), the duration (chronic) and the type (maternal separation) of stress played a role in the turnover of 5-HT in maintaining normal brain function in the PFC. 5. Conclusion These data suggest a close functional relationship between the release of DA from terminals within the PFC and the retrieval of the learning and memory in rats. Overall, early treatment of cognitive impairment-induced exacerbation of Parkinsonism may be a promising strategy to delay a worsening of PD symptoms. These data suggest that DA and 5-HT signaling in the PFC are responsive to FM and may reduce stress-induced cognitive impairment in PD. Acknowledgements The work of the authors was supported by the National Research Foundation of South Africa, the University of KwaZulu-Natal and the College of Health Sciences. We would also like to thank the staff of the Biomedical Resource Centre of the University of KwaZulu-Natal for the technical assistance provided. References Aarsland, D., Kurz, M.W., 2010. The epidemiology of dementia associated with
80
Brain Research Bulletin 132 (2017) 75–81
E. Dallé et al.
Gironell, A., Pagonabarraga, J., Gich, I., 2008. Levodopa and executive performance in Parkinson’s disease: a randomized study. J. Int. Neuropsychol. Soc. 14, 832–841. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates. Academic Press. Pienaar, I.S., Kellaway, L.A., Russell, V.A., Smith, A.D., Stein, D.J., Zigmond, M.J., Daniels, W.M., 2008. Maternal separation exaggerates the toxic effects of 6hydroxydopamine in rats: implications for neurodegenerative disorders. Stress 11, 448–456. Sahin, E., Gumuslu, S., 2004. Alterations in brain antioxidant status: protein oxidation and lipid peroxidation in response to different stress models. Behav. Brain Res. 155, 241–248. Scatton, B., Javoy-Agid, F., Rouquier, L., Dubois, B., Agid, Y., 1983. Reduction of cortical dopamine, noradrenaline, serotonin and their metabolites in Parkinson’s disease. Brain Res. 275, 321–328. Scatton, B., Rouquier, L., Javoy-Agid, F., Agid, Y., 1982. Dopamine deficiency in the cerebral cortex in Parkinson disease. Neurology 32, 1039–1040. Solari, N., Bonito-Oliva, A., Fisone, G., Brambilla, R., 2013. Understanding cognitive deficits in Parkinson’s disease: lessons from preclinical animal models. Learn. Mem. 20, 592–600. Song, L., Che, W., Min-Wei, W., Murakami, Y., Matsumoto, K., 2006. Impairment of the spatial learning and memory induced by learned helplessness and chronic mild stress. Pharmacol. Biochem. Behav. 83, 186–193. Sun, Y.N., Wang, T., Wang, Y., Han, L.N., Li, L.B., Zhang, Y.M., Liu, J., 2015. Activation of 5-HT(1)A receptors in the medial subdivision of the central nucleus of the amygdala produces anxiolytic effects in a rat model of Parkinson’s disease. Neuropharmacology 95, 181–191. Takashima, A., Petersson, K.M., Rutters, F., Tendolkar, I., Jensen, O., Zwarts, M.J., Mcnaughton, B.L., Fernandez, G., 2006. Declarative memory consolidation in humans: a prospective functional magnetic resonance imaging study. Proc. Natl. Acad. Sci. U. S. A. 103, 756–761. Tao, C., Yan, W., Li, Y., Lu, X., 2016. Effect of antidepressants on spatial memory deficit induced by dizocilpine. Psychiatry Res. 244, 266–272. Tata, D.A., Markostamou, I., Ioannidis, A., Gkioka, M., Simeonidou, C., Anogianakis, G., Spandou, E., 2015. Effects of maternal separation on behavior and brain damage in adult rats exposed to neonatal hypoxia-ischemia. Behav. Brain Res. 280, 51–61. Tillerson, J.L., Cohen, A.D., Philhower, J., Miller, G.W., Zigmond, M.J., Schallert, T., 2001. Forced limb-use effects on the behavioral and neurochemical effects of 6hydroxydopamine. J. Neurosci. 21, 4427–4435. Vorhees, C.V., Williams, M.T., 2006. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat. Protoc. 1, 848–858. Williams, S.M., Goldman-Rakic, P.S., 1998. Widespread origin of the primate mesofrontal dopamine system. Cereb. Cortex 8, 321–345. Yamauchi, M., Miyara, T., Matsushima, T., Imanishi, T., 2006. Desensitization of 5-HT2A receptor function by chronic administration of selective serotonin reuptake inhibitors. Brain Res. 1067, 164–169. Yau, J.L., Noble, J., Hibberd, C., Rowe, W.B., Meaney, M.J., Morris, R.G., Seckl, J.R., 2002. Chronic treatment with the antidepressant amitriptyline prevents impairments in water maze learning in aging rats. J. Neurosci. 22, 1436–1442. Yuen, E.Y., Wei, J., Liu, W., Zhong, P., Li, X., Yan, Z., 2012. Repeated stress causes cognitive impairment by suppressing glutamate receptor expression and function of the prefrontal cortex. Neuron 73, 962–977.
Parkinson disease? Neurology 30, 1326–1330. Kulisevsky, J., Garcia-Sanchez, C., Berthier, M.L., Barbanoj, M., Pascual-Sedano, B., Gironell, A., Estevez-Gonzalez, A., 2000. Chronic effects of dopaminergic replacement on cognitive function in Parkinson’s disease: a two-year follow-up study of previously untreated patients. Mov. Disord. 15, 613–626. Liu, J., Wang, X., Mori, A., 1994. Immobilization stress-induced antioxidant defense changes in rat plasma: effect of treatment with reduced glutathione. Int. J. Biochem. 26, 511–517. Luo, X.M., Yuan, S.N., Guan, X.T., Xie, X., Shao, F., Wang, W.W., 2014. Juvenile stress affects anxiety-like behavior and limbic monoamines in adult rats. Physiol. Behav. 135, 7–16. Lupien, S.J., Mcewen, B.S., Gunnar, M.R., Heim, C., 2009. Effects of stress throughout the lifespan on the brain: behaviour and cognition. Nat. Rev. Neurosci. 10, 434–445. Mabandla, M.V., Nyoka, M., Daniels, W.M., 2015. Early use of oleanolic acid provides protection against 6-hydroxydopamine induced dopamine neurodegeneration. Brain Res. 1622, 64–71. Mabandla, M.V., Russell, V.A., 2010. Voluntary exercise reduces the neurotoxic effects of 6-hydroxydopamine in maternally separated rats. Behav. Brain Res. 211, 16–22. Madrigal, J.L., Olivenza, R., Moro, M.A., Lizasoain, I., Lorenzo, P., Rodrigo, J., Leza, J.C., 2001. Glutathione depletion: lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain. Neuropsychopharmacology 24, 420–429. Maeda, T., Kannari, K., Shen, H., Arai, A., Tomiyama, M., Matsunaga, M., Suda, T., 2003. Rapid induction of serotonergic hyperinnervation in the adult rat striatum with extensive dopaminergic denervation. Neurosci. Lett. 343, 17–20. Matsumoto, K., Yobimoto, K., Huong, N.T., Abdel-Fattah, M., Van Hien, T., Watanabe, H., 1999. Psychological stress-induced enhancement of brain lipid peroxidation via nitric oxide systems and its modulation by anxiolytic and anxiogenic drugs in mice. Brain Res. 839, 74–84. Morris, R., 1984. Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47–60. Mpofana, T., Daniels, W.M.U., Mabandla, M.V., 2014. Neuroprotective effects of caffeine on a maternally separated Parkinsonian rat model. J. Behav. Brain Sci. 04 (2), 8. Muck-Seler, D., Pivac, N., Diksic, M., 2012. Acute treatment with fluvoxamine elevates rat brain serotonin synthesis in some terminal regions: an autoradiographic study. Nucl. Med. Biol. 39, 1053–1057. Muller, T., Benz, S., Bornke, C., 2001. Delay of simple reaction time after levodopa intake. Clin. Neurophysiol. 112, 2133–2137. Narayanan, N.S., Land, B.B., Solder, J.E., Deisseroth, K., Dileone, R.J., 2012. Prefrontal D1 dopamine signaling is required for temporal control. Proc. Natl. Acad. Sci. U. S. A. 109, 20726–20731. Narayanan, N.S., Rodnitzky, R.L., Uc, E.Y., 2013. Prefrontal dopamine signaling and cognitive symptoms of Parkinson’s disease. Rev. Neurosci. 24, 267–268. Nikiforuk, A., Popik, P., 2011. Long-lasting cognitive deficit induced by stress is alleviated by acute administration of antidepressants. Psychoneuroendocrinology 36, 28–39. Nowakowska, E., Kus, K., Chodera, A., Rybakowski, J., 2001. Investigating potential anxiolytic: antidepressant and memory enhancing activity of deprenyl. J. Physiol. Pharmacol. 52, 863–873. Pani, L., Porcella, A., Gessa, G.L., 2000. The role of stress in the pathophysiology of the dopaminergic system. Mol. Psychiatry 5, 14–21. Pascual-Sedano, B., Kulisevsky, J., Barbanoj, M., Garcia-Sanchez, C., Campolongo, A.,
81