The effects of citalopram and low-dose risperidone on memory and anxiety in rats subjected to chronic immobilization stress

ABSTRACT OBJECTIVES: Many clinical reports describe the beneficial effects of low-dose atypical antipsychotic added to the antidepressants in the management of anxiety disorders. The aim of this study was to evaluate the effect of low-dose atypical antipsychotic when added to antidepressant treatment on cholinergic M1 receptor expression in the hippocampus and amygdale region in learning and cognitive disorders caused by anxiety. METHODS: The treatments were administered by using different test models on memory, learning, and anxiety, as well as the effect on muscarinic M1 receptor expression levels were assessed. Citalopram (10 mg/kg/sc) and combination [citalopram and risperidone (1 mg/kg/sc)] treatments were applied after stress induction using the immobilization model in rats. Animals groups were randomly divided as: control, stress, stress + citalopram, and stress + combination treatment group. Rats in stress groups were immobilized in cages for 4 h a day for 15 days. On days 1–5, groups were subjected to Morris water maze (MWM), open field, and elevated plus maze (EPM) tests. RESULTS: MWM test results have shown that citalopram induces an anxiolytic effect. Low-dose risperidone treatment has increased the antidepressant-like activity of citalopram in all tests. In OFT the number of squares that rats were circulating on the plane was increased and the time spent by the rats on the maze platform was also increased in MWM. In addition to this, the time spent by the rats on the open arms of the EPM test were also increased. Since the combined treatment increased the discovery of the environment and the active behaviour in tests; all those reflected the increase in general activity. Findings also suggest that treatments may play an effective role in altering the expression level of M1 receptors which are effective in learning and recalling information in the amygdale and the hippocampus. Combination treatment has been shown to provide a meaningful correction of stress-induced memory and learning functions. CONCLUSIONS: These findings indicate that combination treatment may help reduce the stress-induced impairments in cognitive functions.


Introduction
In this study, we hypothesized that muscarinic receptors might have major roles in the recall process of the negative experience during confrontation with the trauma. Consistent with our hypothesis, the role of muscarinic receptors in the production of anxiety has been established in previous studies. These studies showed that either systemic or intracranial injections of the muscarinic M 1 receptor antagonist like pirenzepine could diminish the anxiety in different experimental models [1,2].
Stress is a factor that can cause many neurodegenerative diseases such as anxiety and depression by affecting the neurobehavioural structure of the organism leading to deterioration in the homeostasis of the organism by receiving physical, emotional, mental or social stimuli from the environment of the organism. While the brain has the ability to adapt the changes generated by the stress, on the other hand, it may inadequate to adapt under chronic stress [3].
Since many regions of the brain are constantly in interaction, very few brain functions are associated with a single region. The transmission of the information between the regions through neurotransmitters due to proximity or distance is achieved by a higher complex interaction [4]. Cholinergic and serotonergic systems have an active role in the anxiety biology, while some brain regions are involved in the formation of anxiety [3][4][5]. The serotonergic system projects to the limbic system, which contains amygdale, hippocampus, and cortical structures that are known to be the most important parts involved in memory, moods, and cognition. In addition to this memory processes can be impaired by alterations in the functions of the hippocampus and apparently the frontal cortex and the amygdale under stressful conditions [6]. Besides it is known that muscarinic receptors play major role in memory, cognitive functions, and emotional state [7]. Along with some overlapping factors contributing to the story of anxiety and depression, the symptoms and treatment of these two disorders may be very different. Selective Serotonin Reuptake Inhibitor (SSRI) group antidepressant treatment is effective for both disorders. In these studies, it has been reported that acute or chronic administration of fluoxetine; an SSRI group drug had different effects on anxiety treatment [8,9]. Citalopram (Cit) which belongs to the SSRI group is known to play important role in increasing serotonergic neuronal transmission. Studies have reported that the addition of antipsychotic medications provided efficacy in the treatment of antidepressantresistant anxiety disorder [10,11]. Risperidone (Ris) is a 5-HT 2A , dopamine D 2 , adrenergic α 1, α 2A , and histaminergic H1 receptors blocker which has minimal extra-pyramidal side effects and sedation compared to classical antipsychotics [12,13]. It has been found out that atypical antipsychotic medications used in the treatment of posttraumatic stress disorder (PTSD) alleviate certain symptoms such as arousal and re-experiencing while other symptoms of PTSD do not achieve the same result [14][15][16][17]. In some preclinical and clinical trials, the anxiolytic effect of atypical antipsychotic drugs has been encountered with various results indicating that either they have both an anxiogenic effect or no effect [16,17].
In this current study, the effects of Cit treatment and combination (Cit + Ris) therapy on functions of memory, learning and anxiety were evaluated in rats subjected to chronic immobilization stress with the open field (OFT), elevated plus maze (EPM) and Morris water maze (MWM) tests. The role of muscarinic receptors in anxiety formation has been established in previous studies and the change in the muscarinic acetylcholine M 1 receptor expression levels in amygdale and hippocampus were evaluated in this study.

Test conditions
During the experiments, animals' standard food and water provided ad libitum. In this study, Wistar Albino male rats weighing 250-300 g were used for 8-10 weeks. The rats were kept at 12:12 h light-dark, 21 ± 3°C constant temperature and at 55 ± 5% humidity. The cycles were reversed day and night to obtain active cycles of the rats and adapted the optimum conditions for 1 week in advance. The rats were handled by the same researcher to minimize stress throughout the week for the experimental periods. We used only male rats to avoid female physiological factors since a decreased oestrogen level can be anxiogenic [18]. For this study animal rights were kept in line with the principles of "Guide for the Care and Use of Laboratory Animals, National Research Council (eighth edition)" and necessary ethical permits were taken by Experimental Animal Research Center (MUHDEK approval no: 20.2016.June; DEHAMER, Marmara University, Istanbul).

Experimental groups
The experimental groups and the procedure applied are as follows: were administered before the experiments by being exposed to immobilization for 4 h a day for 15 days.

Immobilization model
Immobilization stress was induced by holding experimental rats in a cylindrical cage surrounded by a wire with 7 cm diameter and 13 cm height for 4 h a day for 15 days.

Morris water maze test
The maze was a circular pool made of Plexiglas (150 cm diameter and 60 cm height), filled with water (30 cm depth) having a temperature of 25 ± 1°C. The maze was made opaque with coloured plastic beads in it. Each rat was located randomly in one of the four quadrants (West, East, North, and South) at the beginning of each trial. A plexiglas platform (diameter of 10 cm, 30 cm in height) was placed 1 cm below the surface of the water. Before learning trials were repeated 3 times, a rat was individually located on the platform for 10 s to adapt it to the task. In four consecutive days of learning experiments, different location (West, East, North, and South) were used every day. Records were taken for 90 s from the rats that left in the water once a day while finding the platform. Rats that were unable to find the platform were kept on the platform and the experiment was repeated again. On the 5th day of the experiment, the platform was removed from the maze and the time spent for locating the platform (escape latency) was scored [19]. During experimental trials, each rat was kept warm under a heating lamp in order not to keep them wet. An elevation in the escape latency period indicated the maze spatial learning, whereas an elevation in correct quadrant time indicated the spatial memory impairment.

Elevated plus maze
The Labyrinth, consists of a central platform (10 cm × 10 cm) made of two open arms (50 cm × 10 cm), two closed arms (50 cm × 10 cm), and black polycarbonate plastic. It is supported by plastic legs 50 cm in height from the ground and both closed arms have 10 cm walls [20].

Open field test (OFT)
The discovery activity of the rats was assessed by an OFT. The test plane is 35 cm in height, 57 cm in width, 76 cm in length with a 48 square grid floor. The rat was placed in one of the corner squares and recorded for 5 min by video camera. Walking time, transitions between area lines and standing movements on the back of two legs (rearing) were evaluated as an activity to discover the environment [22]. In the open field rats, the prolongation of freezing time, the number of circulating squares and the number of rearing behaviour were evaluated as anxiety symptoms according to the control group [23].

Preparation of tissues and immunoblotting
At the end of the 15th day, hippocampus and amygdale were dissected and stored at −80°C according to the Paxinos Rat Brain Atlas of decapitated rats with high dose thiopental sodium (Actavis®,Archimedes Pharma, UK, United Kingdom) s.c. [24]. The tissues were weighed and homogenized by adding a cocktail containing protease inhibitors to 10 mM Tris-HCl (pH 7.2) buffer, homogenized for 90 min at 300×g for 5 min and then at 13,200×g for 90 min. Protein quantities of homogenates obtained were determined by Lowry method and samples of 100 µg were prepared [25]. Samples were run on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis for 1.5 h at 90 V and transferred to a nitrocellulose membrane (Schleicher and Schuell, 0.45 μm, Germany). The membranes were blocked with Tris buffer containing 1% Bovine Serum Albumin and then incubated with the specific M 1 muscarinic receptor primary antibody (1:100) (Santa Cruz Biotechnology Inc, CA, USA) at +4°C for 14 h. Membranes washed at room temperature were incubated with alkaline phosphatase conjugated with secondary antibody (1:1000) for 1 h and the antibody-antigen complex in the membrane was detected by NBT/BCIP (nitro blue tetrazolium and 5bromo-4-chloro-3-indolyl-phosphate; Promega, Wisconsin, USA). For densitometric analysis of membrane, Bio-Rad Molecular Analyst Software (www.totallab. com, Free) was used [26]. The results were standardized using β-actin (Santa Cruz, CA, USA; 1:200). All the other chemicals were obtained from Sigma (St Louis, MO, USA) unless otherwise stated.

Statistical analyses
For statistical analysis GraphPad software (Prism 3.0; GraphPad Software, San Diego, CA, USA) was used. All data were expressed means ± SEM. The groups of data were compared with analysis repeated measures ANOVA followed by Bonferroni multiple comparison post-hoc tests. Statistical significance was accepted to be smaller than p < .05.

Effect of drugs on memory and behavioural experiments
According to the MWM test, the results of each experiment for each group and the comparison of the groups between themselves according to the experimental days were represented in Table 1, starting from Day 1 to Day 5. The experiments starting from day 1 to day 4 were including the platform whereas the experiments on day 5 were not including the platform (Table 1). Platform discovery times were found to vary significantly between day 1 and day 4 when all groups were compared within themselves. The statistical analysis revealed a difference between the control group and all other groups (p < .001). The results obtained in the 5th day when there was no platform were compared regarding to the time spent on the platform area. While the time spent in the platform area in the stress group was significantly increased compared to the control group (p < .001), Cit and combination treatment group showed a significant decrease compared to the stress group (p < .001). There was also a significant difference was found between the combined treatment and Cit groups (p < .01).
According to the EPM scores, in the group receiving immobilization stress, the anxiety index found to be increased significantly compared to the control group (p < .001). As can be seen in Figure 1, both Cit treatment and combination treatment significantly reduced the anxiety index (p < .001, both treatment groups) in the stress group with significantly higher anxiety index.
When the means of the control and stress groups were compared in OFT, the period of freezing time was found to be significantly increased (p < .001, Figure  2(a); the walking time and the transitions between area lines were found to be significantly decreased (p < .001, Figure 2(b) and for the number of rearing behaviour; there was no significant difference was found. Groups with Cit and the combination treatment showed a   decrease in the freezing time (p < .001, for both groups; Figure 2(a) and an increase in the number of transition lines was observed when compared with the stress group (Figure 2(b); p < .01-.001). As can be seen in Figure 2(c), significant difference was found only between the stress group and the groups in which the combination treatment was applied (p < .05), but no significant difference was observed between the other groups.

Immunoblotting analysis
Immunoblotting results obtained by western blot method after homogenization of rat amygdale and hippocampus after dissection were shown in Table 2. The M 1 muscarinic receptor expression levels (p < .05-.01) obtained from amygdale and hippocampus regions of the rats exposed to immobilization stress were found to be decreased compared to the control group. Moreover, according to the stress group; the level of M 1 muscarinic receptor expression in the amygdale was found to be significantly different between the group treated with Cit (p < .05) and the combination therapy (p < .01-.001). The increase in muscarinic M 1 receptor level in the amygdale may suggest the reversal of reduced expression levels due to the stress. As can be seen from Table 2, combination treatment appears to be more effective in reversal of M 1 receptor expression when the effect of treatments on expression level is assessed (p < .001). Similar to the amygdale region; the level of M 1 receptor expression in the control group in the hippocampus was found to be higher than in the stress group (p < .05). Decreased expression level with stress was recovered with treatments. M 1 muscarinic receptor expression in the hippocampus region was compared to Cit and combination treatments groups (p < .05, Figure 3 and Table 2).

Discussion
In this study, possible effects of Cit and the combination treatments on cognitive function and anxiety index were investigated in immobilization stressed rats. It has been shown that learning and memory functions impaired by stress induction are improved at a significant level with combination treatment. In the literature, findings obtained by adding low-dose antipsychotic drugs to anxiety disorder treatment vary. In the recent study, low-dose Ris has been shown to have an antidepressant-like effect in the forced swimming test in mice, but in some studies, it has been shown that there is no anxiogenic effect or even no effect on the symptoms of anxiety [17,[27][28][29]. In our studies for drug effect determination; the used behavioural test model throughout the treatment and the choice of doses were two important factors to be considered. The results of our study, reveals that Cit and combination treatments have changed the level of M 1 muscarinic receptor expression significantly and this suggests that cholinergic system plays an active role in the hippocampus and amygdale regions.
Cholinergic pathways involved in learning and memory functions are projected from the basal forebrain to the cortex and hippocampus. Studies that have investigated the roles of some brain regions in cognitive and emotional processes have shown that the hippocampus is an important brain region for integrating stress-induced cognitive and neurochemical responses [30][31][32][33]. It is known that exposure to chronic stress causes a decrease in the hippocampus and amygdale volume and an increase in amygdale activity. 1.784 ± 0.08 1.498 ± 0.07* 1.762 ± 0.12 + 1.998 ± 0.11 +++,≠ Notes: Control: Applied physiological saline injection group; Stress: Stress induced by immobilization; Stress + Cit: Group after inducing immobilization, treated with citalopram (10 mg/kg); Stress + Cit + Ris: Group after inducing immobilization, treated with citalopram (10 mg/kg) and risperidone (1 mg/kg). The values were represented as mean ± SD. Each group n = 8 were rats. *p < .05 and **p < .01 Comparisons according to control; + p < .05 and ++ p < .01 comparisons according to stress; ≠ p < .05 comparisons according to citalopram treatment (10 mg/kg). Consistent with other studies, our results showed that the group that exposed to the immobilization stress had an increased anxiety index whereas there was a decrease in freezing time and the transitions between area lines. The decrease in anxiety index due to the decrease/deletion of possible negative emotions in the treated groups can be explained by the anxiolytic effect of known SSRI, Cit. In the neuroimaging studies, the activity of the left amygdale decreased at the time when hatred appeared in the face expressions of patients and treated with Cit for seven days where the right amygdale activity is also reduced when the fear expression is seen [34,35]. Although clinical trials in patients with treatment-resistant anxiety disorder have been shown to be effective in the treatment of symptoms following SSRI + antipsychotic combination therapy, the mechanism of their interaction is not fully understood [36][37][38]. Muscarinic receptors play a major role in memory, cognitive functions and emotional state [5]. Studies in experimental animals have indicated that muscarinic receptors are also effective in coding new information and also on learning and short-term memory [26,39]. In anxiety formation studies, using different experimental models have shown that anxiety can be reduced after injection of receptor antagonists such as scopolamine, a muscarinic receptor antagonist. Moreover, it has been shown that scopolamine reduces the release of acetylcholine in the hippocampus and amygdale, causing degradation of spatial memory during coding of information [6,40,41]. The role of muscarinic receptors in many neurodegenerative disorders has been suggested, but the cholinergic mechanisms have not been fully understood in stress condition. A number of studies in the literature indicate that M 1 receptor blockade modulates anxiety rather than M 2 receptors [42,43]. Down-regulation of M 1 receptor expression in the stress group can be seen as a natural consequence of the plasticity established by an effective stress [44]. Our study is shown that both Cit and combination treatment have upregulated M 1 expression in the hippocampus and the amygdale.
The results of our study may show that the effect of Cit in MWM increases the swimming time of the rats and also reduces the freezing time, implying an anxiolytic effect. Moreover, Ris, which is used as an atypical antipsychotic, increases the duration of rats on the platform by reducing the inactivity period of the rats and thus increasing the effect of Cit. The use of the MWM in analysing memory and learning has been reported as has the relationship between performance in the MWM and both drug effects and serotonergic system [45,46]. The results of our study show that the swimming time decreases and the freezing time increases in the immobilization group. These results return to control group levels with citalopram and combined treatments. Consistent with our results recent experimental studies have also shown that risperidone are ameliorated cognitive dysfunction in hippocampus [45,47].
As a result of EPM and OFT tests, it has been found out that Cit and Ris showed an anxiolytic effect. Previous studies have shown that while the combination treatment increases the time spent on the open arms of the EPM, the number of squares circulating in the OFT and the number of rearing; it also decreases the freezing time. In the faces of all these results, combination treatment is thought to increase anxiolytic activity [13,41]. These results were consistent with our results and suggest that the combination treatment is affecting the cholinergic modulation either indirectly or directly through the muscarinic receptors. A report showed that acute administration of SSRIs leads to a reduction in the negative emotions such as fear, anger, and pain where they are replaced by happiness instead. It is known that the increase in happiness in human is explained by the increase in the serotonin level, which is related to the neural processing of social and emotional information. The effect may be due to consecutive increase in the muscarinic M 1 receptors producing a correction of the cognitive functions by setting a new plasticity and learning [26,30]. In previous studies, Cit + Ris antipsychotic effect has been shown to increase extraprimidal side effects. Similarly, the combined use of these two drugs has been reported to have a positive effect on both depression and amnesia models [11,27]. The results of our study may suggest that Cit treatment alone or in combination with low-dose Ris may produce a more potent anxiolytic-like activity.

Conclusions
In conclusion, the use of Cit in combination with Ris showed a positive treatment effect in anxiety with the help of behavioural tests such as OFT, EPM, and MWT and the importance of the M 1 receptor expression level in amygdale and hippocampus was emphasized in anxiety treatment. Our findings emphasize the development of methods for recovering symptoms of stress-induced anxiety and depression, as well as providing answers to behavioural information about selected agents. The results of our study may be potentially useful in helping to better understand the conductional basis of certain drug effects, given the different ways in which anxiety development may affect the learning of the hippocampus and amygdale, which are responsible for cognitive and memory functions. In the face of all these findings, the use of Cit in combination with a low dose of Ris will bring a different perspective to scientific work and may potentially lay the groundwork for the development of alternative treatment regimens in the clinic.

Disclosure statement
No potential conflict of interest was reported by the authors.