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. Author manuscript; available in PMC: 2025 Dec 17.
Published in final edited form as: Neuropharmacology. 2014 Aug 20;86:311–318. doi: 10.1016/j.neuropharm.2014.08.009

Evaluation of 5-HT2A and mGlu2/3 receptors in postmortem prefrontal cortex of subjects with major depressive disorder: Effect of antidepressant treatment

Carolina Muguruza a, Patricia Miranda-Azpiazu a, Rebeca Díez-Alarcia a, Benito Morentin b, Javier González-Maeso c, Luis F Callado a,d,*, J Javier Meana a,d
PMCID: PMC12706166  NIHMSID: NIHMS2115025  PMID: 25150943

Abstract

Several studies have demonstrated alterations in serotonin 5-HT2A (5-HT2AR) and glutamate metabotropic mGlu2 (mGlu2R) receptors in depression, but never in the same sample population. Recently it has been shown that both receptors form a functional receptor heterocomplex that is altered in schizophrenia. The present study evaluates the gene expression and protein density of 5-HT2AR and mGlu2/3R in the postmortem prefrontal cortex of subjects with major depressive disorder (n = 14) compared with control subjects (n = 14) in a paired design.

No significant differences between subjects with depression and controls in the relative mRNA levels of the genes HTR2A, GRM2 and GRM3 were observed. The 5-HT2AR density evaluated by [3H] ketanserin binding was significantly lower in antidepressant-treated subjects (Bmax = 313 ± 17 fmol/mg protein; p < 0.05) compared to controls (Bmax = 360 ± 12 fmol/mg protein) but not in antidepressant-free subjects (Bmax = 394 ± 16 fmol/mg protein; p > 0.05). In rats, chronic treatment with citalopram (10 mg/kg/day) and mirtazapine (5 mg/kg/day) decreased mRNA expression and 5-HT2AR density whereas reboxetine (20 mg/kg/day) modified only mRNA expression. The mGlu2/3R density evaluated by [3H] LY341495 binding was not significantly different between depression and control subjects.

The present results demonstrate no changes in expression and density of both 5-HT2AR and mGlu2/3R in the postmortem prefrontal cortex of subjects with major depressive disorder under basal conditions. However, antidepressant treatment induces a decrease in 5-HT2AR density. This finding suggests that 5-HT2AR down-regulation may be a mechanism for antidepressant effect.

Keywords: Depression, Human brain, mGlu2/3 receptors, 5-HT2A receptors, Antidepressants

1. Introduction

Depression is a severe mental disorder characterized by affective, vegetative, and cognitive symptoms that show a relapsing–remitting course. Within the mental disorders, depression has the highest proportion of total burden and disability across all world regions (Whiteford et al., 2013).

In spite of the thousands of studies carried out, the pathophysiology and etiology of depression remain unknown. Several neurotransmitter systems and functional networks within the brain have been found to be affected in patients with major depressive disorder. Among them, depression has been closely linked to changes in serotonergic neurotransmission, being currently the selective serotonin reuptake inhibitors (SSRIs) the first choice antidepressant treatment (Sharp and Cowen, 2011). Serotonin 5-HT2A receptors (5-HT2A-R) play an important role in mediating the effects of serotonin in many physiologic processes including the modulation of mood and emotion-based actions (Aznar and Klein, 2013). Additionally, these receptors are targets for numerous antidepressants (Artigas, 2013). Several studies have demonstrated alterations in 5HT2A-R density in the brain of depressed subjects (Rosel et al., 2000; Mintun et al., 2004; Rosel et al., 2004; Bhagwagar et al., 2006). However, these studies show contradictory results depending on the brain area studied and the clinical features of the subjects.

More recently, a dysfunction of the glutamatergic system has been also implicated in the pathophysiology of depression (Sanacora et al., 2012). Different studies have provided evidence of reduced glutamate metabolite levels in the frontal cortex of patients with major depressive disorder (Sanacora et al., 2012). The glutamate metabotropic mGlu2 and mGlu3 receptors (mGlu2/3-R) have been also shown to be altered in both animal models of depression and the postmortem brain of subjects with major depressive disorder (Feyissa et al., 2010; Matrisciano et al., 2008). Moreover, both selective mGlu2/3-R agonists and antagonists have shown antidepressant effects in animal models of depression (Chaki et al., 2013).

5-HT2A-R and mGlu2-R form a functional receptor heterocomplex (González-Maeso et al., 2008; Fribourg et al., 2011). Evidence of a ligand binding interaction (Moreno et al., 2012) and a transcriptional cross-regulation (Kurita et al., 2012) between 5-HT2A-R and mGlu2-R has been demonstrated in human brain. Moreover, a dysregulation of this heterocomplex is present in frontal cortex of schizophrenic subjects (González-Maeso et al., 2008; Kurita et al., 2012).

The aim of the present study was to assess the gene expression and protein density of 5-HT2A and mGlu2/3 receptors in the postmortem prefrontal cortex of subjects with major depressive disorder. The study was designed to discriminate the effect of antidepressant treatment on these receptors.

2. Materials and methods

2.1. Postmortem human brain samples

Human brain samples were obtained at autopsies performed in the Basque Institute of Legal Medicine, Bilbao, in compliance with policies of research and ethical boards for postmortem brain studies at the moment of sample obtaining. Deaths were subjected to retrospective searching for previous medical diagnosis and treatment using examiner’s information and records from hospitals and mental health centers. After searching of antemortem information was fulfilled, a total of 14 brains from subjects with diagnosis of major depressive disorder according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) (American Psychiatric Association, 1994) were matched to 14 brains from control subjects in a paired design. Control subjects were chosen among the collected brains on the basis of the absence of neuropsychiatric disorders or drug abuse, and an appropriate gender, age, and postmortem interval (time between death and tissue dissection/freezing) to match each subject in the depression group. Thirteen out of the 14 subjects in the depression group committed suicide. Matched control subjects were mainly death by accidental causes. Therefore, almost all deaths were violent and by sudden mechanisms in both groups. A toxicological screening was performed in the blood of all subjects in order to determine antidepressants, other drugs and ethanol (National Institute of Toxicology, Madrid, Spain). Depression subjects were divided into two groups according to the absence or presence of antidepressant drugs in the blood toxicological screening –termed as antidepressant-free (AD-free, n = 5) and antidepressant-treated (AD-treated, n = 9), respectively. Furthermore, screening and quantification of antidepressant drugs in brain tissue from all subjects with depression was carried out by liquid chromatography–tandem mass spectrometry with a previously validated method (Sampedro et al., 2012). Age, postmortem interval and RIN (RNA integrity number) values were similar in depression and control subjects. Moreover, demographic characteristics did not differ significantly between AD-free and AD-treated subjects with depression (Table 1).

Table 1.

Demographic characteristics of postmortem human brain samples.

Subjects with major depressive disorder (n = 14) and matched controls (n = 14)
Group Gender (M/F) Age (years) PMI (h) RIN
Depression 5M/9F 53.4 ± 4.5 19.2 ± 1.9 6.6 ± 0.4
 AD-F (n = 5) 1M/4F 64.2 ± 8.4 19.8 ± 2.1 6.6 ± 0.5
 AD-T (n = 9) 4M/5F 47.4 ± 4.4 18.9 ± 2.9 6.6 ± 0.4
Control group 5M/9F 53.9 ± 4.6 16.9 ± 2.0 7.2 ± 0.3
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Group values are mean ± SEM. Abbreviations: AD-F (Antidepressant-free), AD-T (Antidepressant-treated), F (female), M (male), PMI (postmortem interval), RIN (RNA integrity number).

Specimens of dorsolateral prefrontal cortex (DLPFC) were dissected at autopsy (0.5–1 g tissue) on an ice-cooled surface following standard procedures (Rajkowska and Goldman-Rakic, 1995), and immediately stored at −80 °C until assays were performed. A full description of demographics of the definitive pairs of AD-free and AD-treated subjects with depression and their individually matched controls can be found in Table S1.

2.2. Animals and treatments

Male Sprague-Dawley rats (SGIKER Animal facilities of the University of the Basque Country, Leioa, Spain) with a final weight of 250–300 g were housed (5 per cage) on a 12 h light/dark cycle at room temperature (22 °C) and 60% humidity with free access to food and water. Animal care and all experimental protocols were performed in agreement with European Union regulations (O.J. of E.C. L276/33, 20/10/2010) and approved by the Animal Welfare Committee.

Rats were injected intraperitoneally with saline (2 ml/kg/day, n = 17), citalopram (10 mg/kg/day, n = 10), mirtazapine (5 mg/kg/day, n = 10) or reboxetine (20 mg/kg/day, n = 8) during 21 days. All drugs were dissolved in saline and daily dosages were administered in two divided doses (one every 12 h). The selected doses of antidepressants were chosen from literature to correspond to clinically relevant treatment (Kugelberg et al., 2001; West et al., 2009; Gould et al., 2003). Rats were killed 48 h after the last injection. Their brains were removed and the cortex dissected and stored at −80 °C until assays were performed. In order to rule out the presence and the acute influence of antidepressant drugs in rat’s brain tissue after chronic treatments, antidepressant brain levels were measured by liquid chromatography-tandem mass spectrometry using a previously validated method (Sampedro et al., 2012).

2.3. Drugs, primers, probes and radioligands

Citalopram and mirtazapine were purchased from Biotrend Chemikalien GmbH (Köln, Germany) and Trocris Bioscience (Bristol, UK), respectively. Reboxetine was donated by Juste S.A.Q.F. (Madrid, Spain).

Primers and probes for the qRT-PCR were provided by Applied Biosystems™ (California, USA) as inventoried TaqMan® gene expression assays. Assay identification numbers for the HTR2A, GRM2, GRM3, GAPDH and ACTB human genes, and Htr2a, Gapdh and Actin rodent genes are specified in Table S2.

[3H]ketanserin (specific activity 67 Ci/mmol) was obtained from PerkinElmer Life and Analytical Sciences and stored at −20 °C. [3H]-2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)-propionic acid ([3H]LY341495; specific activity 40 Ci/mmol) was supplied by American Radiolabeled Chemicals Inc (Missouri, USA) and stored at −20 °C. All other chemicals were obtained from standard sources.

2.4. Quantitative real-time polymerase chain reaction

RNA extraction and purification from human and animal brain samples were performed with the commercial RiboPure™ kit, according to the manufacturer specifications. RNA concentrations were measured in the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, USA) and Brain RNA integrity number (RIN) was assayed using the Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA) as previously reported (Garcia-Sevilla et al., 2010). Reverse transcription was carried out to obtain the complementary DNA (cDNA) with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems™, California, USA).

The mRNA expression of the genes encoding the 5-HT2A, mGlu2 and mGlu3 receptors (HTR2A, GRM2 and GRM3 respectively) was determined by quantitative real-time polymerase chain reaction (qRT-PCR) with a StepOne™ system (Applied Biosystems™, California, USA). For each sample, 20 ng of cDNA in a final volume of 5 µl of reaction mix, containing TaqMan® Fast universal PCR Master Mix and the corresponding pre-designed TaqMan® gene expression assays, were used. The assays identification numbers and characteristics are listed in Table S2. Only one mRNA was amplified in each PCR assay (one gene expression assay per well, singleplex). The mRNA expression amounts of the target genes were normalized to the expression of two housekeeping genes —glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB)— and to the expression of a reference sample included in all PCR plates. Triplicate measurements of each cDNA aliquot were performed. A negative internal control (without cDNA) was also included in all PCR plates for each gene expression assay. The conditions of the qRT-PCR and further details are available in the Supplementary data.

2.5. Radioligand binding assays

2.5.1. Brain membranes preparation

Brain cortex samples (~300 mg wet tissue) were homogenized in 3 ml of ice-cold buffer (5 mM Tris–HCl, 250 mM sucrose, pH 7.4) using an Ultra-Turrax T8 (IKA Labortechnik, Staufen, Germany) at maximum speed for 10 s at 4 °C. The homogenates were centrifuged at 1000 × g for 5 min at 4 °C. The pellets (P1 fractions) were discarded and the supernatant layers re-centrifuged at 40,000 × g for 10 min at 4 °C. The resulting pellets (P2 fractions) were resuspended in 1.5 ml of fresh incubation buffer (50 mM Tris–HCl, pH 7.5 for [3H]ketanserin binding assays and 10 mM potassium phosphate with 100 mM potassium bromide, pH 7.6 for [3H]LY341495 binding assays) and re-centrifuged in similar conditions twice. Protein content was measured according to Bradford’s method (Bradford, 1976) using BSA as standard. Membrane enriched pellets containing 1.2 mg of protein were stored at −80 °C until assay. The day of the experiment, membrane pellets were defrosted (4 °C), thawed and re-suspended in the corresponding incubation buffer to reach a final protein concentration of 0.15 mg/ml approximately. The real final protein content was measured after the experiment according to Bradford’s method.

2.5.2. [3H]Ketanserin and [3H]LY341495 binding assays

Complete saturation binding assays were performed with [3H]ketanserin (0.03–10 nM, 10 concentrations) and [3H]LY341495 (0.07–40 nM, 10 concentrations) in order to determine the density (Bmax) and the affinity (KD) of 5-HT2A and mGlu2/3 receptors respectively. Incubation was carried out in 96-well plates and started with the addition of the membranes (30–40 µg protein per well) in a final incubation volume of 250 µl. Reactions were incubated in a thermostatic shaker (DTS-4 Shaker, ELMI. Riga, Latvia) for 60 min at 37 °C for [3H]ketanserin binding assays and 30 min at 4 °C for [3H]LY341495 binding assays. The presence of methysergide (10 µM) and l-glutamic acid (1 mM) was used to determine the non-specific binding of [3H] ketanserin and [3H]LY341495, respectively. After incubation, free radioligand was separated from bound radioligand by rapid filtration under vacuum (1450 FilterMate Harvester, PerkinElmer) through GF/C glass fiber filters pre-soaked with 0.5% polyethylenimine or incubation buffer. The filters were then rinsed three times with cold incubation buffer, air dried (20 °C, 1.5 h), and counted for radioactivity (5 min) by liquid scintillation spectrometry using a MicroBeta TriLux counter (PerkinElmer) with a counting efficiency of 40–50%. Cases and controls were processed at the same time and all samples were run in duplicate.

2.6. Statistical analysis

The qRT-PCR data analysis was performed in StepOne Software v2.1 (Applied Biosystems TM, California, USA). The relative mRNA amount of each target gene was calculated as 2−ΔΔCt ± SEM —normalized by house-keeping genes expression and reference sample expression— and data were standardize to consider the average of control/saline samples as relative value 1. Data obtained from complete saturation binding experiments with human or rat brain samples were analyzed by non-linear analysis using GraphPad Prism™ software version 5.0. The apparent equilibrium dissociation constant (KD) and the maximum density of specific binding sites (Bmax) were obtained.

Statistical comparisons between depression and control groups were conducted by unpaired Student’s t-test, one-way analysis of variance (ANOVA) followed by a Dunnet’s post-hoc test or by F-test in GraphPad Prism™ software version 5.0. Pearson’s coefficient for simple correlation was calculated to test for possible association among demographic variables (age, postmortem interval and RIN) with relative mRNA expression or with receptors density. The level of significance was chosen as p = 0.05.

Statistical comparisons between rats treated with antidepressants and rats treated with saline were conducted by one-way ANOVA or by F-test in GraphPad Prism™ software version 5.0. The level of significance was chosen as p = 0.05.

All data were subjected previously to a Grubbs’s test in order to determine possible outlier values among experimental groups. The detected outliers were rejected for the statistical analysis.

3. Results

3.1. Gene expression of 5-HT2AR, mGlu2R and mGlu3R in postmortem brain of subjects with depression

The relative mRNA levels of the genes encoding 5-HT2AR (HTR2A), mGlu2R (GRM2) and mGlu3R (GRM3) were evaluated by qRT-PCR in the DLPFC of subjects with depression and matched controls. The mRNA levels did not significantly correlate with the variables age, postmortem interval and RIN either in subjects with depression or in controls for any of the studied genes (data not shown).

No statistically significant differences were found between subjects with depression and controls (Student’s t-test) in the relative mRNA levels of the genes HTR2A (0.89 ± 0.09, p = 0.28), GRM2 (0.96 ± 0.08, p = 0.68) and GRM3 (1.04 ± 0.15, p = 0.84) (Fig. 1A–C). Moreover, when subjects in the depression group were divided according to the presence or absence of antidepressant drugs at time of death, no statistically significant differences were observed between AD-free, AD-treated and control subjects in the relative mRNA levels of any of the studied genes (HTR2A: F [2,24] = 0.86, p = 0.44; GRM2: F[2,24] = 0.37, p = 0.69; GRM3: F [2,25] = 0.36, p = 0.71; in one-way ANOVA followed by a Dunnet’s post-hoc test) (Fig. 1A–C).

Fig. 1.

Fig. 1.

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Relative mRNA levels of the genes HTR2A (A), GRM2 (B) and GRM3 (C) in the DLPFC of subjects with Depression (D, n = 14) —AD-F (antidepressant-free, n = 5) and AD-T (antidepressant-treated, n = 9)— and matched controls (C, n = 14). Bars represent means ± SEM.

3.2. Binding density of 5-HT2AR and mGlu2/3R in postmortem brain of subjects with depression

The density of the 5-HT2AR and mGlu2/3R was evaluated by means of saturation binding assays with the radioligands [3H] ketanserin and [3H]LY341495, respectively, in the postmortem DLPFC of subjects with depression and matched controls.

The saturation binding curves performed with [3H]ketanserin were best fitted to a one site model in both controls (F[2,125] = 0.61, p = 0.54) and subjects with depression (F[2,124] = 0.58, p = 0.56) (Fig. 2A). No statistically significant correlations were found between the maximum binding density (Bmax) of 5-HT2AR and the variables age at death, postmortem interval and RIN neither in subjects with depression nor in matched controls (data not shown). The comparative co-analysis of [3H]ketanserin saturation curves showed no statistically significant differences between depression (Bmax = 346 ± 14 fmol/mg protein; KD = 1.37 ± 0.19 nM) and control subjects (Bmax = 360 ± 12 fmol/mg protein; KD = 1.10 ± 0.13 nM) either in terms of 5-HT2AR Bmax (F[1,253] = 0.58, p = 0.45) or KD (F [1,253] = 1.41, p = 0.24) (Fig. 2A and C). However, when discriminating in regard of the antidepressant treatment, the comparative co-analysis showed statistically significant differences between the Bmax of AD-free, AD-treated and control subjects (F[2,251] = 4.42, p = 0.01; Fig. 2B) without differences in the KD values (F [2,251] = 0.68, p = 0.51; Fig. 2B). Specifically, Dunnet’s comparison post-hoc test over control group, revealed a statistically significant decrease in the 5-HT2AR Bmax in those subjects with depression treated with antidepressant drugs (Bmax = 313 ± 17 fmol/mg protein; F[2,25] = 5.38, p < 0.05) but not in those who were free of antidepressants at time of death (Bmax = 394 ± 16 fmol/mg protein; p > 0.05) (Fig. 2B and C).

Fig. 2.

Fig. 2.

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Saturation curves of [3H]ketanserin specific binding (A, B) and [3H]LY341495 specific binding (D, E) in the DLPC of subjects with Depression (D, n = 14) —AD-F (antidepressant-free, n = 5) and AD-T (antidepressant-treated, n = 9)— and matched controls (n = 14). (C, F) Maximum binding density (Bmax) of 5HT2AR (C) and mGlu2/3R (F) in the DLPC of subjects with Depression (D, n = 14) —AD-F (antidepressant-free, n = 5) and AD-T (antidepressant-treated, n = 9)— and matched controls (n = 14). *p < 0.05 in Dunnett’s Multiple Comparison Test over control group after one-way ANOVA. Bars represent means ± SEM.

On the other hand, the saturation binding curves performed with [3H]LY341495 in order to evaluate the density of the mGlu2/3R were also best fitted to a one site model in both controls (F [2,116] = 0.54, p = 0.58) and subjects with depression (F [2,116] = 1.58, p = 0.21) (Fig. 2D). No statistically significant correlations were found between the Bmax of mGlu2/3R and the variables age at death, postmortem interval and RIN neither in subjects with depression nor in matched controls (data not shown). The comparative co-analysis of [3H]LY341495 saturation binding curves revealed no statistically significant differences between depression (Bmax = 1929 ± 82 fmol/mg protein; KD = 6.62 ± 0.84 nM) and control subjects (Bmax = 2034 ± 81 fmol/mg protein; KD = 5.61 ± 0.69 nM) neither in terms of mGlu2/3R Bmax (F [1,236] = 0.78, p = 0.38) nor KD (F[1,236] = 0.81, p = 0.37) (Fig. 2D and F). When discriminating in regard to the antidepressant treatment, no statistically significant differences were found between the [3H]LY341495 saturation binding curves of AD-free (Bmax = 1970 ± 106 fmol/mg protein; KD = 6.57 ± 1.06 nM), AD-treated (Bmax = 1908 ± 112 fmol/mg protein; KD = 6.65 ± 1.16 nM) and control subjects neither in terms of Bmax (F [2,234] = 0.44, p = 0.65) nor KD (F[2,234] = 0.40, p = 0.67) (Fig. 2E and F).

3.3. Effect of the chronic treatment with antidepressant drugs on gene expression and binding density of 5-HT2AR in rat brain

Due to the 5-HT2AR alterations observed in those subjects who were treated with antidepressant drugs but not in those free of antidepressants, we aimed to evaluate the individual effect of chronic treatment with different antidepressant drugs in the mRNA expression and density of 5-HT2AR.

After a 21-day chronic treatment with the antidepressants citalopram (10 mg/kg/day), mirtazapine (5 mg/kg/day) and reboxetine (20 mg/kg/day), and a 48-h washout period, the liquid chromatography-tandem mass spectrometric analyses in rat’s brain tissue gave negative results for all the antidepressant drugs. Therefore, we were able to discard the acute presence of antidepressant drugs in rat’s brain tissue after chronic treatments and in turn the possible influence of these drugs in the in-vitro assays.

Gene expression assays showed a significant decrease in the relative mRNA levels of Htr2A gene in the brain cortex of the three groups of rats treated with antidepressants —citalopram (−30.5 ± 7.0%, p < 0.01), mirtazapine (−27.8 ± 7.7%, p < 0.05) and reboxetine (−33.6 ± 7.3%, p < 0.01) compared with those rats treated with saline (100 ± 5.7%; F[3,37] = 6.06, p = 0.002) (Fig. 3A).

Fig. 3.

Fig. 3.

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Relative mRNA levels of the gene HTR2A (A) and saturation curves of [3H] ketanserin specific binding (B) of 5HT2AR in the brain cortex of rats chronically treated with citalopram (n = 10), mirtazapine (n = 10), reboxetine (n = 8) or saline (n = 17). *p < 0.05 and **p < 0.01 in Dunnett’s Multiple Comparison Test over control group after one-way ANOVA. Bars represent means ± SEM.

Saturation curves of [3H]ketanserin binding in rat’s brain cortex were best fitted to one-site model in all groups of treatment (saline: F[2,165] = 0.21, p = 0.81; citalopram: F[2,94] = 0.04, p = 0.96; mirtazapine: F[2,94] = 0.69, p = 0.50; reboxetine: F[2,75] = 0.28, p = 0.76) (Table 2, Fig. 3B). The co-analysis of the curves showed statistically significant differences among groups in terms of Bmax (F [3,437] = 14.26, p < 0.001) without differences in the KD (F [3,437] = 0.89, p = 0.45) (Table 2, Fig. 3B). The Dunnett’s post-hoc test revealed a statistically significant decrease in the maximum binding density of 5-HT2AR in the brain cortex of those rats treated with citalopram (p < 0.01) and mirtazapine (p < 0.001) compared with the saline group (F[3,41] = 36.8, p < 0.001; Table 2, Fig. 3B). No statistically significant differences were found for the rats treated with reboxetine compared to saline treated rats (Table 2, Fig. 3B).

Table 2.

Parameters of [3H]ketanserin saturation binding curves in brain cortex of rats chronically treated with antidepressants or saline.

Group Bmax (fmol/mg protein) KD (nM)
Saline (n = 17) 472.7 ± 19.0 1.10 ± 0.15
Citalopram (n = 10) 376.2 ± 17.9** 0.93 ± 0.16
Mirtazapine (n = 10) 238.7 ± 12.2*** 1.60 ± 0.26
Reboxetine (n = 8) 513.5 ± 22.7 1.10 ± 0.17
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**

p < 0.01,

***

p < 0.001, in Dunnett’s Multiple Comparison test over control group (saline) after one-way ANOVA. All values are mean ± SEM.

4. Discussion

The present results show the absence of differences between subjects with depression and controls either in mRNA levels of the genes encoding 5-HT2AR, mGlu2R and mGlu3R or in the protein radioligand density of these receptors. Conversely, a decrease in the 5-HT2AR density in those subjects with depression treated with antidepressant drugs is demonstrated.

Several studies have assessed 5-HT2AR in the brain of subjects with depression showing different results depending on the brain area studied. For the present study we chose the DLPFC. Changes in this brain area have been consistently identified in resting, cognitive activation and treatment imaging studies performed in patients with depression (Fitzgerald et al., 2006). Previous postmortem studies have reported mainly no significant differences in 5-HT2AR gene expression and binding density in the prefrontal cortex of subjects with depression when compared with control subjects (Stockmeier et al., 1997; Rosel et al., 2000; Marazziti et al., 2003; Rosel et al., 2004; López-Figueroa et al., 2004; Sibille et al., 2004; Oquendo et al., 2006). Our results confirm that 5-HT2AR seem to be not altered in treatment-free depressed subjects. In this sense, different in vivo positron emission tomography (PET) studies have also reported unaltered 5-HT2AR binding in the cortex of antidepressant-free depressed patients compared to healthy controls (Meyer et al., 1999; Meltzer et al., 1999; Meyer et al., 2001; Mintun et al., 2004; Sheline et al., 2004). However, other PET studies have also reported both decreases (Biver et al., 1997; Attar-Levy et al., 1999; Yatham et al., 2000) and increases (Bhagwagar et al., 2006) of 5-HT2AR binding in the brain cortex of unmedicated patients with depression. The reason for these discrepancies may be related to the clinical characteristics of the patients or to methodological issues. Thus, the interpretation of results from in vivo imaging studies may be complicated by factors such ligand specificity and modeling (Bhagwagar et al., 2006). In fact, PET neuroreceptor imaging studies do not distinguish between changes in receptor density or affinity (Bhagwagar et al., 2006).

Together with the effects of treatment, potential explanations for discrepancies with other reports are also parameters intrinsic to the studies carried out in postmortem human brain tissue samples such as age, inclusion of control subjects that are not individually matched by demographic variables, and the reliability of postmortem psychiatric diagnosis mostly based on postmortem family interviews.

Another confounding factor in postmortem studies is suicide. It is known that the mortality risk for suicide is increased in subjects with depression (Hawton & van Heeringen, 2009). In the present study, 13 out of the 14 subjects included died by suicide. Several reports have demonstrated an increase in 5-HT2R density in the prefrontal cortex of suicide victims (Mann et al., 1986; Arango et al., 1990; Turecki et al., 1999; Pandey et al., 2002). It is unclear whether this finding is associated with a specific diagnosis as depression or represents a direct feature of suicide subjects. In fact, this changes seems to be more common in studies of suicide victims who died by violent methods. Moreover, higher postmortem prefrontal 5-HT2R binding has also been shown to correlate with lifetime aggression in suicide (Oquendo et al., 2006). Nevertheless, other studies have not found significant differences in prefrontal cortex 5-HT2AR density neither between suicide and non-suicide subjects (Oquendo et al., 2006) nor between depressed suicide subjects and controls (Owen et al., 1983; Crow et al., 1984; Cheetham et al., 1988; Arranz et al., 1994; Lowther et al., 1994; Stockmeier et al., 1997; Rosel et al., 2000; Underwood et al., 2012). Additionally, we have previously reported that prefrontal cortex [3H]ketanserin binding did not differ in a group of suicide victims with a variety of psychiatric disorders as compared to controls (Muguruza et al., 2013).

A further element to analyze in studies related to subjects with depression is the effect of the pharmacological treatment. The present study was designed in a paired way, differentiating between antidepressant-free and antidepressant-treated subjects. Our results showed that treatment with antidepressant drugs reduced the 5-HT2AR density in the prefrontal cortex of subjects with depression without affecting its mRNA expression. In this sense, it has been previously demonstrated that depressed patients showed a significant decrease in 5-HT2AR binding in several cortical regions following desipramine, clomipramine and paroxetine treatments (Yatham et al., 1999; Attar-Levy et al., 1999; Meyer et al., 2001). In the same way, the neuroimaging studies in which subjects recently received antidepressant treatment usually reported decreased 5-HT2AR density, whereas no change was found in those with no recent antidepressant use (Meyer, 2008). The chronic administration of antidepressants to rats in the present study confirmed that despite the three used antidepressants decreased the HTR2A mRNA levels in the rat brain cortex, only citalopram and mirtazapine induced a statistically significant decrease in the 5-HT2AR density. It has been suggested that 5-HT2AR oppose the therapeutic effects of activating non-5-HT2AR in depression (Marek et al., 2003). Thus, the decrease in 5-HT2AR density in the prefrontal cortex of subjects with major depressive disorder induced by the antidepressant treatment might contribute to the clinical efficacy of these drugs. In fact, mirtazapine would augment the clinical response to SSRIs in treatment-resistant patients by blocking 5-HT2AR (Marek et al., 2003). The blockade of 5-HT2AR might enhance in cortical brain areas the neurotransmission mediated by 5-HT1AR, an effect that has been linked to antidepressant efficacy (Artigas, 2013). It is also known that 5-HT2AR density has an inverse relationship to extracellular serotonin levels such that binding will decrease when serotonin is chronically increased by antidepressants like SSRIs. The possible mechanisms for the 5-HT2AR down-regulation induced by antidepressant treatment include an increase in receptor degradation or changes in gene transcription as here reported (Gray and Roth, 2001). In this sense, the present results evidenced that in the prefrontal cortex of subjects with major depressive disorder these mechanisms of 5-HT2AR down-regulation induced by antidepressant drugs might take place at the post-transcriptional level, as opposed to what occurs in rat’s brain cortex. In any case, an acute effect of the antidepressant treatment can be discarded in our animal experiments because a 48 h wash-out period was used.

A previous study has reported a significant increase in mGlu2/3R immunoreactivity in total homogenates from postmortem prefrontal cortex samples of depressed subjects (Feyissa et al., 2010). Conversely, mGlu3R mRNA expression was down-regulated among suicides with and without major depressive disorder in two areas of the prefrontal cortex (Sequeira et al., 2009). The differences with the present results may be due to the different techniques used to measure mGlu2/3R, the different sample preparation or to the distinct cortical brain areas assessed. In fact, the radioligand [3H] LY341495 binds both subtypes of group II mGluR and there is not currently any specific radioligand to discriminate between both populations. So that, the results obtained by means of radioligand binding or non subtype-specific antibodies could be masking possible independent alterations for each of the receptor subtypes. Regarding differences between brain areas, spontaneously depressed rats showed a reduced expression of mGlu2/3R in the hippocampus with no changes in cerebral cortex (Matrisciano et al., 2008). Additionally, mGlu2/3R expression has been reported to be reduced in mouse hippocampus in an animal model of depression (Wierońska et al., 2008). This decrease was reversed after antidepressant administration (Wierońska et al., 2008). We found no significant alterations in neither the mRNA expression or in the protein density of mGlu2/3R in the prefrontal cortex of depressed subjects compared to controls regardless of the antidepressant treatment. In line with these results, other studies performed in rodents and non-human primates have also reported the absence of alterations of cortical mGlu2/3R after a chronic treatment with antidepressant drugs (Pałucha et al., 2007; Feyissa et al., 2010). However, accumulating evidence from preclinical studies highlight the potential of mGlu2/3R ligands as antidepressant drugs (Chaki et al., 2013) or as adjunctive drugs in the treatment of depression (Matrisciano et al., 2007). In this context it has been suggested that the antidepressant effect of the mGlu2/3R selective antagonists may be mediated by an increase in glutamate release or by modulation of the prefrontal dopaminergic system (Ago et al., 2013; Chaki et al., 2013). A recent study has also demonstrated that GRM3 polymorphisms do not contribute to genetic susceptibility to depression (Jia et al., 2014).

5. Conclusions

In conclusion, the present results demonstrate no changes in expression and density of both 5-HT2AR and mGlu2/3R in the postmortem prefrontal cortex of subjects with major depressive disorder under basal (antidepressant-free) conditions. However, a decrease in 5-HT2AR density in the prefrontal cortex of depressed subjects treated with antidepressants is shown. This finding suggests that 5-HT2AR down-regulation may be a mechanism involved in antidepressant effect.

Supplementary Material

Supplementary tables S1-S3

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.neuropharm.2014.08.009.

Acknowledgments

This work was supported by the Basque Government (IT616-13 and S-PE12UN033), the University of the Basque Country (UFI 11/35), Ministry of Science (SAF2009-08460), Ministry of Economy and Competitiveness (SAF2013-48586-R), NIH (5R01MH084894) and the Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM, Spain. CM and PMA were recipients of predoctoral fellowships from the University of the Basque Country. The authors wish to thank the staff members of the Basque Institute of Legal Medicine, Bilbao for their cooperation in the study, the University of the Basque Country’s SGIKER for animal and analytical facilities, and Cecilio Álamo (JUSTE, S.A.Q.F., Madrid) for the generous providing of reboxetine.

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Supplementary tables S1-S3
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