Introduction

Depression is one of the neuropsychiatric diseases that most affect people's daily lives. It is a mood disorder that negatively affects daily activities such as emotions, thoughts, sleeping, eating or working [1]. The causes of depression are not yet fully understood, but a number of psychological, environmental and biological factors are known to play a role in the development and course of the disease [2]. The presence of some of the symptoms can be described as operational, such as anorexia and weight loss, sleep disturbances, cognitive and psychomotor changes and therefore can be evaluated in laboratory animals [1].

It is believed that stress is the most important factor in the etiology of depression [3]. Chronic exposure to stressful life events is a risk factor for major depression [4]. Environmental factors and stress factors can affect biological systems by causing excessive glucocorticoid release or axial irregularity in the hypothalamo-pituitary axis (HPA), especially in the physiological response to stress. In addition to depressive symptoms, it can also cause changes in the limbic system and cortical brain areas [5]. In rodents, chronic unpredictable stress or chronic mild stress has been found to increase behavioral hopelessness and cognitive impairment. These effects are likely mediated by HPA hyperactivity and increased corticosterone secretion [4]. Under stressful conditions, the hypothalamus plays an important role in the response to psychosocial stress by regulating the secretion of the corticotropin releasing factor (CRF). The paraventricular nucleus (PVN) of the hypothalamus secretes CRF, which stimulates the release of adrenocorticotropic hormone (ACTH) by the anterior pituitary. ACTH then stimulates glucocorticoid release by the adrenal glands. Glucocorticoids have a wide range of metabolic effects in the body [6].

It is known that the HPA axis, which is a junction between central and peripheral pathways, also plays a key role in the pathogenesis of mood disorders. In rodents, exogenous corticosterone has been used to induce anxiety and depression-like changes in behavior, neurochemistry and brain morphology [7, 8]. Various behavioral, neurochemical, neuroendocrine and neuroimmune changes similar to those in human depression have been observed in rats as a result of chronic mild stress (CMS) model procedures [1]. Therefore, the CMS model developed by Willner et al. largely satisfies the various validity criteria desired for an animal depression model [1, 9]. Exposure to CMS increases the duration of immobility of rats in the forced swim test (FST). Reversal of these effects with chronic antidepressant treatment makes the chronic mild stress model the most valid model of depression [10]. Symptoms of depression caused by CMS can be reversed with typical antidepressants such as tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs), serotonin norepinephrine reuptake inhibitors (SNRIs), monoamine oxidase inhibitors (MAOIs) [11].

Serotonin (5-HT) is a neurotransmitter that plays a role in many physiological processes in the central nervous system (CNS), including mood regulation, circadian rhythm (sleep-wakefulness), or cognition [12]. 5-HT plays an important role in neuronal and synaptic plasticity by making a neurogenic effect in CNS [3]. 5-HT has been reported to modulate neuroplasticity, especially in early life. 5-HT catalyzes the maturation of astroglia cells and affects the migration of neurons. Synaptic plasticity is a well-known key mechanism in learning and memory [13]. Sertraline is the most commonly used SSRI antidepressant in the treatment of depression, anxiety and obsessive–compulsive disorder [14, 15]. Sertraline prevents the reuptake of 5-HT from the synaptic space in the CNS and allows the accumulation of 5-HT in the synaptic cleft. As a result, sertraline shows its effect by increasing the 5-HT level in the CNS [16, 17].

Neurogenesis is the process of producing new neurons from neural stem cells or progenitor cells in the brain throughout life, although it is most active in the prenatal period and decreases with age in adulthood. Neural stem cell and progenitor cells are called neural precursor cells. There are two neurogenic niche regions in the adult brain. The first is located in the subventricular zone (SVZ) of the lateral, ventricle; the second is in the subgranular zone (SGZ) of the hippocampus. Recently, it has been shown that neurogenesis continues in adulthood in the hypothalamus, which regulates peripheral signals for food intake and energy balance [18].

The neural precursor cells of the hypothalamus are called tanycytes [19, 20]. Hypothalamic diagnoses are special ependymal glial cells lining the third ventricle. Tanycytes can regenerate and allow the formation of new neurons after birth [21]. Tanycytes are divided into 4 groups according to localization and gene expression differences; α1, α2, β1 and β2. The α2 and β1 tanycytes are located on the lateral wall of the third ventricle. It is associated with anorexigenic and orexigenic neurons. β2 tanycytes, on the other hand, occupy the floor of the third ventricle and are in contact with the cerebrospinal fluid-median eminence barrier [18]. Tanycytes are nutrient and metabolite sensors that affect plasticity and neuronal function in the hypothalamus. It plays an important role in energy intake and expenditure [22]. Tanycytes penetrate into the hypothalamic parenchyma and come into contact with arcuate nucleus (AN) neurons involved in the regulation of food intake. In this way, tanycytes perceive and respond to changes in energy balance [23]. Factors controlling the proliferation and differentiation of specific hypothalamic neural stem cell types, including tanycytes, are thought to be of great importance in understanding body weight control [23]. Newly formed neurons in the adult hypothalamus display a typical phenotype of hypothalamic neural circuits and express neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons associated with food intake [18].

The swimming behavior analyzed in the FST is related to the 5-HT system. SSRI group antidepressants cause an increase in swimming behavior. In addition, depression-like behaviors in FST in chronic mild stress can also occur as a result of morphological and/or neurochemical changes in the hippocampus, amygdala and prefrontal cortex. Conditions such as cell loss, decrease in neurogenesis and increase in dendritic atrophy are effective in the neuropathogenesis of depression [24].

Different studies have been conducted to reveal the pathophysiology of depression and to develop new treatment methods. Animal studies show that exposure to chronic stress reduces hippocampal neurogenesis and that sertraline, an SSRI antidepressant, increases adult hippocampal neurogenesis and neurotrophic factor expression, and can improve the effect of stress on hippocampal atrophy. However, no study examining the effect of sertraline on neurogenesis occurring in the hypothalamus, which is the main center of stress and food intake regulation, has not been found in the literature.

In this study, we aimed to determine the possible effects of sertraline, on the expression levels of some molecules associated with neurogenesis and food intake in the hypothalamus in rats with depression with a CMS.

Materıals and Methods

Ethics Statement

The protocols of animal experiments were approved by the Local Ethics Committee of Application and Research Center of Experimental Medicine, Necmettin Erbakan University, No. 2018-020, on 18.05.2018.

Experimental Protocol

Animals

56 adult (6 months old) male Wistar albino rats were used for this study. Animals were taken care of in Necmettin Erbakan University Experimental Medicine Application and Research Center. Rats were housed in plastic cages where they could move freely with food and water containers, their food and water were given as ad-libitum. Cages were cleaned weekly. The animals were stored at the room temperature of 22 ± 1 °C for 12 h light/dark period under standard laboratory conditions (Fig. 1).

Fig. 1
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Schematic representation of experimental protocol

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The animals were divided into 4 groups:

  1. (1)

    Control group (n = 14, C): Solvent (DMSO) was administered subcutaneously with an osmotic minipump.

  2. (2)

    Depression group (n = 14, D): Depression was induced in animals by applying the CMS protocol. Solvent (DMSO) was administered subcutaneously with an osmotic minipump.

  3. (3)

    Depression + Sertraline (n = 14, DS): Sertraline (10 mg/kg/day) was administered subcutaneously with the CMS protocol and osmotic minipump.

  4. (4)

    Sertraline group (n = 14, S): Sertraline (10 mg/kg/day) was administered subcutaneously with an osmotic minipump.

Preparation of Sertraline

For S and DS groups, 10 mg/kg/day sertraline dissolved in 1 ml DMSO was administered (Sigma-Aldrich, USA). Solvent DMSO infusion was applied to control animals. All drug solutions were freshly prepared on the day of the experiment.

Drug Administration

A combination of ketamine (100 mg/kg) and xylazine (5 mg/kg) was administered to all rats intraperitoneally for the insertion of osmotic minipumps and anesthetized. A pouch was opened to accommodate the pump by blunt dissection by opening 0.8–1 cm on the dorsal areas of the animals, and osmotic minipumps (Alzet 2002) were placed subcutaneously. In the application of sertraline groups, sertraline dissolved in DMSO was placed in the osmotic minipump reservoir for 15 days, with sertraline infusion at a dose of 10 mg/kg/day. DMSO, the solvent of sertraline, was administered to the control and depression groups.

Weight Tracking

On the 0 and 31st day of the experiment, all animals were weighed and weight follow-ups were made. % increase/decrease rates of weight changes were calculated.

Chronic Mild Stress (CMS) Protocol

The CMS protocol used was a modification of the one proposed by Wilner [8]. In CMS applications, various stressors were applied to animals for 28 days (Table 1). The animals were also exposed to various stressors at different times to prevent them from getting used to and guessing. All stressors were applied for at least two weeks and the cycle was repeated. In wet cage application, 333 g of wood chips in a cage were wetted with 1.5 l of water. The rats were kept on wet wood chips for 7 h, starting at different times in the morning. In the inclined cage application, the cage with the animals was given an angle of 60 degrees and the animals were kept in this way for 7 h, keeping the food part of the cage in the upper part. In noise stress, a ringing tone of about 60 db intensity was applied for 4 h. Swimming stress was floated for 10 min in plexiglass cylinder vessels (25 cm diameter and 60 cm height, water level 39 cm) where FST was performed. They were taken to the drying apparatus and dried. For hunger stress, only water containers were left in the cages of the animals, the animals were fasted for 16 h after their feed was collected in the cages. The rats were kept in the room for 16 h (04.00 pm–08.00 am), leaving the light of the room where the rats were located under constant lighting stress. The restraint stress was placed in a restraint apparatus with front vent holes made of plexiglass, which could be adjusted to their size, and held for 45 min. [8, 25].

Table 1 Chronic mild model experiment scheme
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Behavioral Tests

Open field test (OFT), forced swimming test (FST) and elevated plus maze test (EPM) were applied to evaluate the locomotor activity, anxiety/depression-like behaviors of the animals. OFT was held between 09:00 and 12:00 am. FST and EPM were applied between 01:00 and 05:00 pm. On the days when more than one behavioral test was performed on the same day, a period of 2 h was left between tests. The animals were brought to the room where the test would be performed at least one hour before the test and they were adapted to the environment.

Open Field Test (OFT)

In order to evaluate the locomotor activities of the animals, before starting the experiments, an OFT was carried out on the 15th day and at the end of the experiment. OFT apparatus used is made of 80 × 80 × 30 cm square black plexiglass material. All test applications were recorded with the video recording system associated with the special software program (Ethovision Video Monitoring System XT11, The Netherlands). With software program, two regions are determined in the test apparatus, center and edge. The parameters of the distance (cm) and speed (cm/s) for 5 min were calculated with a software program. The test apparatus was cleaned with 10% ethyl alcohol solution after each animal [26].

Forced Swimming Test (FST)

FST was used to evaluate depression-like behaviors. Cylindrical plexiglass apparatuses with a diameter of 25 cm and a height of 50 cm were used in the FST. These apparatuses were filled with water at a temperature of 25° up to a height of 40 cm. FST was carried out in two stages as pretest and test. In the pre-test, rats were floated for 15 min and then dried and returned to their cages. 24 h after the pretest, the animals were left one by one in the FST apparatus and recorded with a video recording system for 5 min. The waters were changed after each animal. In this 5-min period, the immobility time, swimming time and climbing time values of the animals were calculated [27].

Elevated Plus Maze Test (EPM)

The EPM apparatus is made of a black plexiglass material, consists of two opposite open arms (50 × 10 cm) at a height of 50 cm from the ground, two closed arms (50 × 10 cm) and a central area (10 × 10 cm) where the arms meet. The closed arms are open at the top and closed at the sides of 40 cm in height. Regions were selected through the software program on the EPM apparatus, and the time (s) the animal spent in the open arms, closed arms and the center within the test period of 5 min, the number of entries to open arms and closed arms were determined. With these values, the percentage of time spent in open arms, which is widely used for anxiety assessment, was calculated. The animals were left in the apparatus in the central area, facing the open arm, and between tests, the test apparatus was cleaned with a 10% ethyl alcohol solution [28].

Ending the Experiment and Removing Tissues

After completing the behavioral tests, the animals were decapitated under anesthesia and their hypothalamus was removed on dry ice according to the Paxinos and Watson rat brain atlas. It was placed in cryotubes and immediately frozen in liquid nitrogen. All tissues were stored at − 80 °C until molecular analysis.

Gene Expression Analysis

RNA isolation was performed from hypothalamus tissues with organic Trizol method to evaluate expression at gene level. The concentration and quality of total RNA samples were checked by spectrophotometric and agarose gel electrophoresis method. All samples were found to be of a quality that can be used in qRT-PCR analysis (Fig. 2). DNAse-I enzyme reaction was performed according to the manufacturer's instructions in order to eliminate possible gDNA contamination. Genes used in quantitative Real-time PCR (qRT-PCR) analysis and primers of reference (PGK1 and CycA) genes were designed using the IDT PrimerQuest (https://eu.idtdna.com/site) program or taken from the literature (Table 2). cDNA was synthesized from the RNA samples that were quality controlled using the manufacturer's protocol (Bio-Rad iScriptTM cDNA Synthesis Kit#170-8891, USA). Expression quantitation analysis of target and reference genes was performed using a real time PCR device (Bio-Rad CFX Connect Real Time PCR System). In order to confirm that the products obtained from real-time PCR are the right products, the products were run and observed in 2% agarose gel at 120 V for 30 min (Fig. 3). In Melting curve (MC) analysis, it was determined that all PCR products were specific and no other genome region was oxidized (Fig. 4).

Fig. 2
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Sample total RNA gel electrophoresis of hypothalamus tissues

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Table 2 Primary sequences of genes used in qPCR assays
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Fig. 3
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Gel image of qRT-PCR products of genes used in the study. M; 100 bp DNA marker

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Fig. 4
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Melting curve (MC) analysis curves of the genes used in the study

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Determination of Serum Corticosterone Levels

Serum corticosterone levels were analszed according to the method of Tintos [30]. Corticosterone-3-(Ocarboxymethyl) oxime (Cortisol-3-CMO), Cortisol-3-CMO ester, and Cortisol-3-CMO-BSA were prepared according to the methods of Tintos [30]. All chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany) unless otherwise stated. Briefly, corticosterone-BSA stock (1 ug/ml) was diluted with a carbonate buffer (pH 9.6) and was added (200 µl/well) into a 96-well microtiter plate (Nunc, Roskilde, Denmark). The plate was incubated overnight at + 4 °C and then washed with wash buffer using an eight-channel pipette. The binding sites not occupied by the coating antigen were then blocked by the blocking buffer (2 g BSA/100 ml PBS pH 7.2, 200 µl/well) for 2 h at 37 °C. The plate was washed again and serum samples (50 µl/well) or standards (50 µl/well) were pre-incubated with primary (50 µl/well) antibodies for 45 min at 37 °C. They were then transferred into coated plates for competition with antigens on the solid phase for 30 min at 37 °C. After washing, biotinylated anti-rabbit antibody (goat antirabbit IgG) was added (100 µl/well), and the plate was incubated for 30 min at 37 °C. Following washing, streptavidin peroxidase solution (100 µl/well, Sigma-Aldrich, Taufkirchen, Germany) was added, and the plate was incubated for 15 min at 37 °C. Tetramethylbenzidine substrate (150 µl/well) was added following washing, and the plate was incubated in the dark for 10 min. Stop solution (sulfuric acid 10%, 50 µl/ well) was added, and the absorbance was measured at 450 nm using a microplate reader (Biotek, Synergy HT, Winooski, USA). The dynamic range of the assays was 10–2000 ng/ml. Inter- and intra-assay coefficients of variations were below 10%.

Statistical Analysis

Data analysis of the study was carried out with SAS University Edition 9.4 program. Descriptive statistics about variables are given. Arithmetic Mean ± Standard Error (AM ± SE) for numerical variables, frequency and percentage values for qualitative variables were given. p < 0.05, p < 0.01, p < 0.0001 were considered significant. one-way ANOVA or mixed effect model was used in the analysis of numerical variables. Tukey test was performed for multiple comparisons. In the analysis of gene expression data, firstly, Ct values expressing the expression levels of all genes subject to the study were normalized with Ct values of PGK1 and CycA reference genes and 2(−∆Ct) values were determined. 2(−∆Ct) values were compared between groups by one-way analysis of variance. Standard error plots of the mean squares of the relevant variables were drawn. In order to compare gene expression levels between control and treatment group, fold change and 2−∆∆Ct statistics is often preferred. In our study, however, there are four groups and we would like to compare each groups to define any physiological differences. As suggested by Livak and Schmittgen (2001), 2−∆Ct values were calculated for each group as ∆Ct = Ct (gene of interest) − Ct (housekeeping gene) and used in variation analysis [31].

Results

Body Weight

Animal weights (g) measured at the beginning of the CMS applications and the last day of the experiments were compared. While a statistically significant decrease was observed in the D group between the last and first measured values in animal weights, an increase was observed in the other groups (p < 0,01), (Table 3; Fig. 5).

Table 3 Body weight in groups (gr)
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Fig. 5
figure 5

Weight change values of control (C), depression (D), sertraline (S) and depression + sertraline (DS) groups at the beginning (0. day) and at the end (30th day) of the CMS applications (**p < 0.01). Compared to day 0 values of each group were evaluated using ANOVA

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Open Field Test

At the beginning of the experiment, on the 15th day and on the 30th day of the experiment, the distance moved, the speed and the percentage of movement parameters were analyzed in the OFT.

Distance Moved Parameter

The distance moved parameter showed a significant difference between the 3 measurement values with respect to time (p < 0.0001), between groups (p < 0.01) and group time interaction (p < 0.01). According to the first distance moved parameter of the groups, changes were found in the second and third measurement values. A significant increase was observed in the sertraline group compared to all other groups at the 30th day measurements (p < 0.01) (Table 4; Fig. 6).

Table 4 Open field test findings
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Fig. 6
figure 6

Distance moved parameters in the OFT. When the three measurements were compared in the experiment, the distance moved at the end of the experiment was lower in all groups. A significant increase was observed in the sertraline group compared to all other groups in measurements on the 30th day (**p < 0.01)

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Velocity Parameter

For speed values, there was a significant difference between the 3 measurement values according to time (p < 0.0001), between groups (p < 0.01) and group time interaction (p < 0.01). A statistically significant decrease was observed in the second and third measurement values according to the first velocity parameter of the groups. A significant increase was observed in the sertraline group compared to the other groups at the 30th day measurements (p < 0.01) (Table 4; Fig. 7).

Fig. 7
figure 7

In the open field test, a significant difference was found between the three measurement values of the velocity parameter (**p < 0.01). When the three measurements were compared in the experiment, the rate was lower in all groups at the end of the experiment. A significant increase was observed in the sertraline group compared to the other groups at the 30th day measurements (**p < 0.01)

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Movement Parameter

In the open field test, the movement percentage changes of the animals showed a statistically significant difference between the groups in the analysis of the measurements made at the beginning of the experiment, on the 15th day and at the end of the experiment (p < 0.01). It was observed that the values of the groups decreased in the second and third measurements according to the first movement percentages. A significant increase was observed in the sertraline group compared to the other groups at the 30th day measurements (p < 0.01) (Table 4; Fig. 8).

Fig. 8
figure 8

In the open field test, the movement percentage changes of the animals showed a statistically significant difference between the groups in the analysis of the measurements made at the beginning of the experiment, on the 15th day and at the end of the experiment (**p < 0.01). A significant increase was observed in the sertraline group compared to the other groups at the 30th day measurements (**p < 0.01)

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Elevated Plus Maze Test

The elevated plus maze test was applied to all groups at the end of their experiments. Percentage values of time spent by the animals in open arms were calculated. When this value was compared between groups, a significant increase was observed in sertraline group compared to all other groups (p < 0.01) (Table 5; Fig. 9).

Table 5 The percentage of time spent in the open arms value in elevated plus maze test (%)
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Fig. 9
figure 9

Percentage of time that the control (C), depression (D), sertraline (S), and depression + sertraline (DS) groups spent on the open arms in the elevated plus maze test at the end of their experiment, compared to the other groups (**p < 0.01)

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Forced Swimming Test (FST)

In the FST, on the 15th and 30th days of chronic stress applications, the immobility time, climbing time, swimming time and movement percentage parameters of the depression group and the control group were determined and compared between the groups.

Immobility Time

In the forced swimming test, the immobility time of the animals on the 15th day of the experiment and in the analysis of the measurements made at the end of the experiment showed a statistically significant difference according to time (p < 0.0001), between groups (p < 0.0001) and in group time interaction (p < 0.0001). Groups D and DS were the groups with the highest immobility times (Table 6; Fig. 10).

Table 6 Forced swimming test findings
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Fig. 10
figure 10

In the Forced Swimming Test, on the 15th day of the experiment, the immobility time of the C and S groups was significantly lower than the D groups (****p < 0.0001). On the 30th day of the experiment, the immobility time of the D group was significantly higher than the DS group (****p < 0.0001)

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Swimming Time

In the forced swimming test, the swimming time of the animals showed a statistically significant difference in the analysis of the measurements on the 15th day of the experiment and on the 30th day of the experiment according to time (p < 0.0001), between groups (p < 0.0001) and in group time interaction (p < 0.05). Depression group animals showed the least swimming behavior (Table 6; Fig. 11).

Fig. 11
figure 11

In the Forced Swimming Test, on the 15th day of the experiment, swimming times in groups C and S were significantly higher than in groups D (*p < 0.05). On the 30th day of the experiment, the swimming time of the D group was significantly lower than the C, S and DS groups (****p < 0.0001, ***p < 0.001)

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Climbing Time

In the forced swimming test, the climbing times of the animals showed a statistically significant difference in the analysis of the measurements on the 15th day of the experiment and on the 30th day of the experiment according to time (p < 0.05), between groups (p < 0.001) and in group time interaction (p < 0.0001). Depression group animals showed the least climbing behavior (Table 6; Fig. 12).

Fig. 12
figure 12

In the forced swimming test, on the 15th day of the experiment, the climbing times in the C and S groups were significantly higher than in the D Groups (*p < 0.05). On the 30th day of the experiment, the climbing time of group D was significantly lower than that of group S (****p < 0.0001)

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Movement Parameter

In the forced swimming test, the movement percentage changes of the animals showed a statistically significant difference between the groups (p < 0.0001) according to the analysis of the measurements performed on the 15th day of the experiment and on the 30th day of the experiment (Table 6; Fig. 13).

Fig. 13
figure 13

In the Forced Swimming Test, on the 15th day of the experiment, the percentage of movement in the C and S groups were significantly higher than in the D groups (****p < 0.0001). On the 30th day of the experiment, the percent movement value of the DS group was significantly higher than the D group (****p < 0.0001)

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Gene Expression Analysis Findings

IGF1R, FGF2, FGFR1, FGFR2, GLUT2, POMC and NPY gene expressions 2(−ΔCt) in the hypothalamus tissues were evaluated and calculated by one-way analysis of variance.

GLUT2 Gene Expression Level in the Hypothalamus

When GLUT2 gene expression levels were examined in the hypothalamus tissue, a statistically significant decrease was observed in group D compared to other groups (p < 0.05), (Table 7; Fig. 14).

Table 7 Gene expression analysis findings
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Fig. 14
figure 14

GLUT2 expression levels in hypothalamus tissues belonging to control (C), depression (D), sertraline (S) and depression + sertraline (DS) groups. Gene expression 2(−∆Ct) levels were compared by one-way analysis of variance (*p < 0.05) (compared with the other group)

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FGF2 Gene Expression Level in the Hypothalamus

There was no statistically significant difference between the groups in terms of FGF2 gene expression levels in the hypothalamus tissue (p > 0.05) (Table 7; Fig. 15).

Fig. 15
figure 15

FGF2 expression levels in hypothalamus tissues belonging to control (C), depression (D), sertraline (S) and depression + sertraline (DS) groups. Gene expression 2(−∆Ct) levels were compared with one-way analysis of variance (p > 0.05)

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FGFR1 Gene Expression Level in the Hypothalamus

There was no statistically significant difference between the groups in terms of FGFR1 gene expression levels in hypothalamus tissue (p > 0.05) (Table 7; Fig. 16).

Fig. 16
figure 16

FGFR1 expression levels in hypothalamus tissues belonging to control (C), depression (D), sertraline (S) and depression + sertraline (DS) groups. Gene expression 2(−∆Ct) levels were compared by one-way analysis of variance (p > 0.05)

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FGFR2 Gene Expression Level in the Hypothalamus

When the gene expression levels of FGFR2 gene expression levels in the hypothalamus tissue were examined compared to the C, DS and S groups of the D group, a statistically significant increase was found in the depression group (*p < 0.05) (Table 7; Fig. 17).

Fig. 17
figure 17

FGFR2 expression levels in hypothalamus tissues belonging to control (C), depression (D), sertraline (S) and depression + sertraline (DS) groups. Gene expression 2(−∆Ct) levels were compared by one-way analysis of variance (*p < 0.05) (comparison of the depression group with other groups)

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IGF1R Gene Expression Level in the Hypothalamus

There was no statistically significant difference in IGF1R gene expression levels between the groups (p > 0.05). However, a statistically insignificant increase was observed in the expression of IGF1R in the DS group (p = 0.064) (Table 7; Fig. 18).

Fig. 18
figure 18

IGF1R expression levels in hypothalamus tissues belonging to control (C), depression (D), sertraline (S) and depression + sertraline (DS) groups. Gene expression 2(−∆Ct) levels were compared with one-way analysis of variance (p > 0.05)

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BDNF Gene Expression Level in the Hypothalamus

There was no statistically significant difference in BDNF gene expression levels between the groups (p > 0.05). However, a statistically insignificant increase in BDNF expression was observed in the DS and S group compared to the D group (p = 0.094) (Table 7; Fig. 19).

Fig. 19
figure 19

BDNF expression levels in hypothalamus tissues belonging to control (C), depression (D), sertraline (S) and depression + sertraline (DS) groups. Gene expression 2(−∆Ct) levels were compared with one-way analysis of variance (p > 0.05)

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POMC Gene Expression Level in the Hypothalamus

A statistically significant increase was found in the POMC gene expression levels of the D group compared to the C, DS and S groups (p < 0.05), (Table 7; Fig. 20).

Fig. 20
figure 20

POMC expression levels in hypothalamus tissues belonging to control (C), depression (D), sertraline (S) and depression + sertraline (DS) groups. Gene expression 2(−∆Ct) levels were compared by one-way analysis of variance (*p < 0.05) (comparison of the depression group with other groups)

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NPY Gene Expression Level in the Hypothalamus

The NPY gene expression level in the hypothalamus tissue was found to be a statistically significant decrease in the depression group compared to the control group (p < 0.05) (Table 7; Fig. 21).

Fig. 21
figure 21

NPY expression levels in hypothalamus tissues belonging to control (C), depression (D), sertraline (S) and depression + sertraline (DS) groups. Gene expression 2(−∆Ct) levels were compared by one-way analysis of variance (*p < 0.05), (control comparison of the depression group)

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Determination of Serum Corticosterone Levels

Figure 22.

Fig. 22
figure 22

Serum corticosterone levels control (C), depression (D), sertraline (S) and depression + sertraline (DS) groups. The serum corticosterone levels were higher in the depression group (****p < 0.0001)

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Discussion

Depression is the most common psychiatric disorder affecting more than 350 million people around the world [32]. The pathogenesis of depression is not fully understood [33]. A common chronic mental disorder, mainly manifests itself with changes in emotion, cognition, behavior, sleep, and appetite, and may cause impairment in social functions [32, 33]. Among mental disorders, the most comprehensive research has been conducted on depression and its treatment. The reason for this is that depression is the most common psychiatric disorder in the world, as well as the serious and vital complications it carries with it. The dramatic increase in the use of antidepressant drugs throughout the world in the last 20–25 years is another proof of this. In addition, according to the data of the World Health Organization (WHO), depression ranks fourth in the world in terms of burden of disease [34, 35].

Chronic mild stress in rodents is an approved and widely used model of depression. The model involves exposing rodents to mild and unpredictable daily stress factors (oblique cage, food or water deprivation, wet cage, continuous light, loud noise, etc.). The protocol consists of mild and non-traumatic everyday stress factors and has been adopted as a method close to modeling the human condition [8, 36, 37].

5-HT shapes neuronal networks and its deficiency fundamentally affects the pathophysiology of mood and mental disorders during the development of neurons [38]. At the same time, many studies testing the effects of stress on 5-HT neuronal activity and 5-HT1A autoreceptor function in experimental animals with a chronic mild stress model have shown that spontaneous 5-HT activity can change dramatically in brain regions [4].

It has been reported that chronic treatment of depression-like behaviors induced by the CMS model with SSRIs desensitizes 5-HT1A autoreceptors and reverses stress-induced behavior [4]. Beyond known neurochemical mechanisms, SSRIs have been reported to induce developmental plasticity due to 5-HT. Increased neuronal plasticity has been noted as a result of high 5-HT levels after treatment [13]. 5-HT modulates glutamatergic transmission and can stimulate N-methyl-D-aspartate receptor-dependent plasticity. 5-HT binds to cell adhesion molecules that are part of the extracellular matrix and are crucial for developmental plasticity. 5-HT increases the polysialized form of the neural cell adhesion molecule (PSA-NCAM), which plays a role in synaptogenesis and neuron remodeling [13, 38].

Malberg and his friends [39] reported that the number of newborn cells increased as a result of 14 and 28 days of antidepressant drug administration. Another study has shown that 5-HT has an overall positive regulatory effect on both the formation of neural precursor cells and the survival of newly produced neurons [40].

In our study, OFT for locomotor activity, EPM test for anxiety, and FST behavioral tests to evaluate depression were used to investigate the effectiveness of SSRI group antidepressants in animals and normal animals with CMS model with depression.

Weight Change Assessment

Experimental animal studies have shown that exposure to chronic stress can cause depression-like symptoms such as anhedonia, disturbed sleep patterns, decreased interest in sexual activity, and significant changes in weight [2, 41].

It has been reported that in animals exposed to chronic stress, weight gain is suppressed during the stress factor and the weight of stressed animals increases during the recovery phase [2]. Food intake was also reported to be reduced in rats with depression with the restriction stress model, and as a result, body weight decreased [42]. [41]. In our study, a statistically significant decrease was observed in the depression group when animal weights were compared at the beginning of the experiments and the last day of the experiments, which supports the findings in the literature, while an increase was observed in the other groups.

Evaluation of Behavior Tests

In a study, an increase in spontaneous activity was reported, especially in the first minute of the test, when rats that were depressed with a CMS model were released into a new environment. It has also been reported that rats tend to have higher activity in the middle squares in the OFT. This may represent a pattern of psychomotor agitation observed in some depressed people [43].

In another study, it was shown that physical stress (repeated mild foot shocks) causes immobility, while emotional stress (witnessing foot shocks) causes hyperactivity [44]. In the present study, OFT was performed to test the locomotor activities of animals. In the OFT, the speed, distance moved, percentage of movement and parameters of the animals were evaluated. While there was no significant difference between the groups at the beginning of the experiment in the parameters of distance moved, speed and percentage of movement of the animals, a statistically significant increase was observed in the sertraline group compared to the other groups in the analysis at the end of the experiment.

At the end of the experiment, it was observed that the animals in the depression group had higher parameters of speed, distance moved, and percentage of movement in the OFT compared to the animals in the control group. This suggests that psychomotor agitation may cause hyperactivity in animals, as stated in the literature. There is no clear explanation for such spontaneous fluctuations observed in other studies [43].

It has been reported that rats with depression induced by the CMS model have increased open arm entry in the EPM test, increased the time spent in the closed arm and exhibit anxiety-like behaviors, and it has been reported that SSRI group antidepressants can reduce these anxiolytic effects [42]. The elevated plus maze is a rodent model of anxiety. No change in the prevalence of extreme anxiety-like and avoidant behaviors was observed in EPM in control and chronic mild stress model animals. Animals not exposed to the chronic mild stress model and treated with sertraline spent more time in the open arm than control and stressed animals. According to these results, animals did not enter into anxiety with the stress model we applied.

In a previous study, administration of sertraline has been shown to reduce immobility and increase swimming behavior in experimental animals, and the results obtained confirm previous findings that sertraline has antidepressant-like properties [45]. In literature, it has been shown that the CMS model increases behavioral hopelessness and cognitive impairment [4]. A significant increase in the time of inactivity in the FST has been reported in mice exposed to four weeks of stress [41]. In our study, a significant increase in immobility time was observed in animals exposed to chronic stress for four weeks compared to the control group. In rats in which depression was induced by the CMS model, both the CMS model continued for 2 weeks and treatment with sertraline (10 mg/kg/day) was administered. Compared to the D group animals, a significant reduction in the duration of immobility was observed in the DS group animals. A statistically significant increase was observed in the swimming behavior of the animals in the DS group and the S group compared to the other groups. The data obtained support evidence that SSRI therapy reverses depression-like behaviors and anxiogenic effects induced by CMS [4, 8, 32].

Depressive disorder involves emotional, cognitive, autonomic and endocrine alterations and also evidences support the role of stress in the development of this disorder. The hypothalamic–pituitary–adrenal axis is involved in the stress response with a concomitant rise in plasma corticoids [46]. After 21 days of chronic stress, significant changes were found in corticosterone levels following antidepressant treatment [47]. In this study, we observed that corticosterone level was significantly higher in the serum of the rats depression groups. A statistically significant decrease in corticosterone level was also observed in the sertraline administered depression group (Fig. 22). Our findings support earlier reports which there is a relationship between high corticosterone levels and depression.

Evaluation of Gene Expression Analysis

It is thought that the stress-induced decrease in neurogenesis may be one of the most important causes of depression [40]. Chronic antidepressant treatment increases the division and proliferation of neural progenitor cells, differentiation, maturation and integration rate of precursor cells [48].

Brain-Derived Neurotrophic Factor (BDNF) is one of the key factors affected in mood disorders. It has been suggested that atrophy associated with depression and neuronal cell death may be important factors mediating the decrease of BDNF expression in the hippocampus and PFC [14, 49]. In some studies, conflicting findings have emerged regarding BDNF gene expression analyzes in animals in which depression was induced by the CMS model. Chiba and his friends [42] reported that BDNF level did not change in PFC with chronic mild post-stress antidepressant treatment, and also that CMS did not have a significant effect on BDNF expression in the hippocampus. They reported down-regulation of BDNF in the PFC, hippocampus, and dentate gyrus after CMS [36]. It is thought that this inconsistency may be due to methodological differences such as the duration of CMS, the post-stress recovery time, the stress procedure, and the method of detection [42]. In this study, in which we used the experimental CMS model, no decrease was observed in the BDNF level in the hypothalamus tissue of the rats in the depression group, while the BDNF levels of the fourteen-day sertraline treatment increased with depression, although it was not statistically significant.

Since insulin-like growth factor-1 (IGF-1) affects many brain processes such as tissue differentiation, synaptic flexibility, and adult neurogenesis, it has been suggested that changes in IGF-1 may play a role in the development of emotional disorders [50]. Chronic antidepressant drug therapy has been shown to increase IGF-1 and IGF-1R expression and phosphorylation [51]. In our current study, it was observed that IGF-1R gene expression level decreased in the D group, but increased in the DS group, although it was not statistically significant.

Fibroblast Growth Factor 2 (FGF2) mediates the growth, differentiation and protection of progenitor cells in neuronal development [52, 53]. FGF2 promotes the growth of axonal branches without affecting the elongation of axons, resulting in increased complexity of axonal trees. FGF2 has been found to be the most effective growth factor in axonal branching [54]. FGF2 signaling has been reported to be required for α-tanycytes proliferation. FGF2 binds to FGFR1 and FGFR2 as receptors. It has been shown that the activation of FGFR1 and FGFR2 can stimulate endothelial cell migration and invasion [55]. In the present study, FGF2, FGFR1 and FGFR2 gene expression levels were investigated in hypothalamus tissue. No statistically significant difference was found between the groups in terms of FGF2 and FGFR1 gene expression levels. A statistically significant increase in FGFR2 gene expression was observed in the Depression group compared to the other groups. The observation of weight loss in depressed animals suggests that more studies are needed to understand the relationship of the FGFR2 receptor to food intake.

The localization areas of the tanycytes can be followed with neuronal stem cell markers. It has been reported that dividing cells migrate to the parenchyma of the hypothalamus when they mature, differentiating into neurons expressing NPY, AgRP or POMC. It has also been reported that neurogenesis regulated by tanycytes plays a role in the regulation of energy balance. Tanycytes express the insulin-independent glucose transporter GLUT2 [22].

The hypothalamic nuclei trigger cellular responses that alter feeding behavior and peripheral glucose homeostasis by integrating the environmental signal. Thus, it maintains the balance between energy consumption and food intake. One of the most important signals controlling this process is glucose. However, the exact mechanism of detection of this molecule is not known [56,57,58].

It has been shown that GLUT2 to be found in third ventricle neurons, astrocytes, endothelial cells and tanycytes, which are involved in AN. Therefore, it is thought that the regulation of orexigenic and anorexigenic peptide expression by GLUT2-dependent sensors can be indirectly controlled [59]. In a new study, it has been reported that glucose-sensing neurons bound to GLUT2 send projections to NPY and POMC neurons [60]. It has been reported that the suppression of GLUT2 can only interrupt the normal glucose sensing process and cause reduced food intake [61]. GLUT2 has been reported to be found especially in the brain nuclei belonging to the limbic system. This particular anatomical distribution suggests that GLUT2 may be associated with a particular neurotransmitter system. It is thought that this system may be the serotonergic system with an anatomical distribution similar to GLUT2 and defined as an important neurotransmitter in the regulation of food intake [62]. In the present study, a significant decrease in the GLUT2 gene expression level was observed in depressed experimental animals. This decrease was reversed with sertraline treatment. According to the information in the literature, it is thought that the decrease in GLUT2 expression level causes a decrease in food intake and may lead to a decrease in weight gain. There is evidence that GLUT2-dependent glucose sensors are critical physiological regulators of nutrition. Although the location of sensors connected to GLUT2 has been identified, its mechanism is still unclear [59]. GLUT2 is thought to be a potential drug target to treat metabolic diseases associated with nutritional disorders [63].

Changes in body weight and feeding behavior were observed in experimental animals with restraint stress. In a study conducted in mice, it has been shown that chronic restraint stress causes hyperactivity of POMC neurons in hypothalamic AN and decreased activity of dopamine neurons in ventral tegmental area [64]. In our study, it was observed that the POMC gene expression level in the hypothalamus was increased in animals with depression. Decreased NPY expression has been reported in the hypothalamus of experimental animals after repeated exposure to stress. Some studies have shown that NPY expression increases, decreases or does not change depending on the type and length of stress exposure. It has been reported that NPY gene expression in the amygdala, which is an important brain region for fear and anxiety, increases after foot shock stress, does not change with acute water avoidance stress, but decreases with acute restraint stress. As a result of these findings, it has been shown that the type of stressor has a great effect on the NPY system in the brain region [65]. In the present study, a statistically significant decrease was observed in the NPY gene expression level of the group in which depression was created by exposure to repeated stress compared to the other groups.

Conclusion

Experimental animal models that accurately imitate the stressors causing mood disorders in human are very helpful in understanding the pathology. However, the neuronal circuitry underlying stress-induced changes in mood and energy balance is not fully understood. In this study, Sertraline reversed depressive symptoms in behavioral parameters. It has been determined that experimental depression may have a negative effect on neurogenesis, and sertraline may reverse this effect. Exposure to depression may cause anorexigenic effect in the hypothalamus, while antidepressant administration may have an orexigenic effect and also play a role in glucose transport in the hypothalamus. Further researches are needed to understand the relationship between depression and food intake better.