Introduction
Major depressive is the most prevalent mental disorder with an estimated prevalence of 13.5–21.2% (Reference Kessler and Walters1–Reference Kessler, Berglund, Demler, Jin, Merikans and Walters3). It is believed that 5% of the population suffers from depression (Reference Murphy, Laird, Monson, Sobol and Leighton4). Together with schizophrenia, depression is responsible for 60% of suicides worldwide and it is predicted to be the second main cause of disability in 2020, regardless of the age and the gender of the patient (5).
The pathophysiological mechanisms and the pharmacological treatment of major depressive disorder focus mainly on the monoamine hypothesis (Reference Skolnick6). This hypothesis predicts that the major depression results from a dysregulation of neurotransmission by serotonin, norepinephrine and dopamine, and the treatments are based on normalising the reduced levels of these neurotransmitters (Reference Charney7). In fact, almost all clinically used antidepressants increase the extracellular concentrations of serotonin or norepinephrine by inhibiting their reuptake from the synapse or by blocking their degradation by inhibiting monoamine oxidase activity (Reference Castrén8,Reference Shelton9). Furthermore, monoamine-based antidepressants remain the first line of therapy for depression, but their long therapeutic delays and low remission rates (about 30%) have encouraged the search for more effective agents (Reference Krishnan and Nestler10).
The treatment of depression was revolutionised by the discovery of monoamine oxidase inhibitors and tricyclic antidepressants. Since then, the availability of newer drugs with less adverse effects has greatly increased the ability to safely treat a significant number of patients (Reference Zarate, Singh and Carlson11). Although commonly used antidepressants, such as the selective serotonin (5-HT) reuptake inhibitor (SSRI), are often effective, full efficacy is only apparent after several weeks, and many patients only partially respond (Reference Millan12–Reference Morilak and Frazer14).
Paroxetine is functionally classified as an SSRI, which enhances serotonergic transmission by blocking the pre-synaptic active membrane transport mechanism for the reuptake of serotonin and consequently increases serotonergic activity at the post-synaptic receptor (Reference Johnson15,Reference Richelson16). Nortriptyline is a metabolite of amitriptyline with several putative pharmacological mechanisms including blockade of norepinephrine and serotonin uptake, blockade of sodium channels and sympathetic blockade and antagonism of N-methyl-d-aspartate glutamate receptors (Reference Richelson16). Venlafaxine is used as the inhibitor of both serotonin and norepinephrine. The primary function of venlafaxine is to protect the transport of serotonin and norepinephrine at the synapse, thus increasing the concentration of both monoamines within the synapse (Reference Duman, Heninger and Nestler17).
Tissues with high-energy demands, such as the brain, contain a large number of mitochondria, being therefore more susceptible to the reduction of aerobic metabolism. Mitochondria are intracellular organelles that play a crucial role in adenosine triphosphate (ATP) production (Reference Calabrese, Scapagnini, Giuffrida-Stella, Bates and Clark18). Most cell energy is obtained through oxidative phosphorylation, a process requiring the action of various enzyme complexes located in the inner mitochondrial membrane, that is the mitochondrial respiratory chain (Reference Horn and Barrientos19). Mitochondrial dysfunction has been shown to be involved in the pathogenesis of a number of diseases affecting the brain, such as dementia, cerebral ischemia, Alzheimer's disease and Parkinson's disease (Reference Blass20–Reference Moreira, Santos, Seiça and Oliveira26). In this context, several recent works also support the hypothesis that energy impairment is involved in the pathophysiology of depression (Reference Tretter, Mayer-Takacs and Adam-Vizi27–Reference Stanyer, Jorgensen, Hori, Clark and Heales30).
Therefore, on the basis of the hypothesis that energy impairment may be involved in the pathophysiology of depression, in the present work, we evaluated the activities of the mitochondrial respiratory chain complexes in the brain of rats and submitted the chronic administration of paroxetine, nortriptyline and venlafaxine.
Materials and methods
Animals
Adult and male Wistar rats (250–300 g) were obtained from Central Animal House of the Universidade do Extremo Sul Catarinense. They were caged in group of five with free access to food and water and were maintained on a 12-h light–dark cycle (lights on 07:00), at a temperature of 23 ± 1°C. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior recommendations for animal care, with the approval of the Ethics Committee from Universidade do Extremo Sul Catarinense.
Drugs
Animals received daily intra-peritoneal injections of paroxetine (10 mg/kg), nortriptyline (15 mg/kg) or venlafaxine (10 mg/kg) in 1.0 ml/kg volume for 15 days (n = 6 animals per group). All drugs were dissolved in saline solution (vehicle). Control animals received the vehicle (1.0 ml/kg). The selection of this regimen was based on previous studies showing important neurochemical and antidepressant effects for both the drugs (Reference Nestler, McMahon, Sabban, Tallman and Duman31–Reference Ide, Fujiwara and Fujiwara35).
Tissue and homogenate preparation
Twelve hours after the last injection, the rats were killed by decapitation, the brain was removed and the prefrontal cortex, hippocampus, striatum, cerebellum and cerebral cortex were homogenised (1:10, w/v) in SETH buffer, pH 7.4 (250 mM sucrose, 2 mM EDTA (ethylene diamine tetraacetic acid), 10 mM Trizma base, 50 IU/ml heparin). The homogenates were centrifuged at 800 × g for 10 min and the supernatants kept at −70°C until used for enzymes activity determination. The maximal period between homogenate preparation and enzyme analysis was always less than five days. Protein content was determined by the method described by Lowry and colleagues (Reference Lowry, Rosebough, Farr and Randall36) using bovine serum albumin as the standard.
Activities of mitochondrial respiratory chain enzymes
NADH (nicotinamide adenine dinucleotide) dehydrogenase (complex I) was evaluated by the method described by Cassina and Radi (Reference Cassina and Radi37) by the rate of NADH-dependent ferricyanide reduction at λ = 420 nm. The activities of succinate-2,6-dichloroindophenol (DCIP)-oxidoreductase (complex II) and succinate:cytochrome c oxido-reductase (complex II–III) were determined by the method described by Fischer and colleagues (Reference Fischer, Ruitenbeek and Berden38). Complex II activity was measured by following the decrease in absorbance because of the reduction of 2,6-DCIP at λ = 600 nm. Complex II–III activity was measured by cytochrome c reduction from succinate at λ = 550 nm. The activity of cytochrome c oxidase (complex IV) was assayed according to the method described by Rustin and colleagues (Reference Rustin, Chretien and Bourgeron39), measured by following the decrease in absorbance because of the oxidation of previously reduced cytochrome c at λ = 550 nm. The activities of the mitochondrial respiratory chain complexes were calculated as nanomole per minute milligram protein.
Statistical analysis
Data were analysed by Student's t-test when F was significant. All analyses were performed using the Statistical Package for the Social Science (SPSS) software.
Results
It was first investigated the respiratory chain complex activities in the presence of nortriptyline in homogenates from prefrontal cortex, hippocampus, striatum, cerebellum and cerebral cortex from rat brain. Figure 1 shows that rats administered with this antidepressant presented an increase in complex II activity in hippocampus and striatum, whereas prefrontal cortex, cerebellum and cerebral cortex were not affected (Fig. 1b). Furthermore, complex IV activity was increased in prefrontal cortex, striatum and cerebral cortex, without affecting cerebellum and hippocampus (Fig. 1d). On the other hand, chronic administration of nortriptyline did not affect complex I and II–III activities in the tested cerebral structures (Fig. 1a and c, respectively).
Fig. 1. Effects of nortriptyline chronic administration on mitochondrial respiratory chain complex I activity (a), complex II activity (b), complex II–III activity (c) and complex IV activity (d) in the prefrontal cortex, cerebellum, hippocampus, striatum and cerebral cortex of rats (n = 6). *p < 0.01 versus saline group, according to ANOVA followed by the Student's t-test.
The next set of experiments was performed in order to evaluate the effect of paroxetine on the respiratory chain complex activities. Figure 2 shows that chronic administration of paroxetine increased complex I activity in prefrontal cortex, hippocampus, striatum and cerebral cortex, while cerebellum was not affected (Fig. 2a). In addition, complex II activity was increased by this antidepressant in hippocampus, striatum and cerebral cortex (Fig. 2b) and complex IV activity was increased in prefrontal cortex (Fig. 2d), with no effect on other brain structures. In contrast, complex II–III activity was not altered by paroxetine administration in either tested structure (Fig. 2c).
Fig. 2. Effects of paroxetine chronic administration on mitochondrial respiratory chain complex I activity (a), complex II activity (b), complex II–III activity (c) and complex IV activity (d) in the prefrontal cortex, cerebellum, hippocampus, striatum and cerebral cortex of rats (n = 6). *p < 0.01 versus saline group, according to ANOVA followed by the Student's t-test.
Finally, we tested the influence of chronic administration of venlafaxine on the respiratory chain complexes activities. It can be seen in Fig. 3 that complex II activity was increased in hippocampus, striatum and cerebral cortex of rats administered with venlafaxine (Fig. 3b), while complex IV activity was increased only in prefrontal cortex (Fig. 3d). On the other hand, respiratory chain complex I and II–III activities were not altered by paroxetine administration in either tested structures (Fig. 3a and c, respectively).
Fig. 3. Effects of venlafaxine chronic administration on mitochondrial respiratory chain complex I activity (a), complex II activity (b), complex II–III activity (c) and complex IV activity (d) in the prefrontal cortex, cerebellum, hippocampus, striatum and cerebral cortex of rats (n = 6). *p < 0.01 versus saline group, according to ANOVA followed by the Student's t-test.
Discussion
Mitochondrial oxidative phosphorylation is the major ATP-producing pathway, which supplies up to 95% of the total energy requirement in the cells (Reference Rex, Schickert and Fink40). In most organisms, the mitochondrial respiratory chain is composed of four enzyme complexes, where electron transport drives translocation of protons from the mitochondrial matrix to the inter-membrane space. The dissipation of this proton gradient generated through ATP synthase catalyses the formation of ATP by the phosphorylation of ADP (adenosine diphosphate) (Reference Boekema and Braun41).
Damage to the mitochondrial electron transport chain has been suggested to play an important factor in the pathogenesis of some psychiatric disorders (Reference Fattal, Budur, Vaughan and Franco42,Reference Madrigal, Olivenza and Moro43), including major depression. Indeed, Gardner and colleagues (Reference Gardner, Johansson and Wibom44) showed a significant decrease of mitochondrial ATP production rates and mitochondrial enzyme ratios in the muscle of major depressive disorder patients. Considering that life stressors may contribute to the development of depression, chronic stress has been used as an animal model of depression. In this scenario, it has been reported that brain Na+, K+-ATPase and respiratory chain complexes I, III and IV activities are inhibited after chronic variate stress in rats (Reference Gamaro, Streck, Matté, Prediger, Wyse and Dalmaz45,Reference Rezin, Cardoso and Gonçalves46) and that complexes I–III and II–III of mitochondrial respiratory chain are inhibited in the rat brain after chronic stress (Reference Madrigal, Olivenza and Moro43). Assis and colleagues (Reference Assis, Rezin and Comim47) also reported that acute administration of ketamine and imipramine increased creatine kinase activity in the brain of rats.
In the present study, we observed that chronic administration of nortriptyline, paroxetine and venlafaxine increased respiratory chain complexes activities in the prefrontal cortex, hippocampus, striatum and cerebral cortex of rats. Our data are in agreement with previous works showing that chronic administration of paroxetine modulate brain energy metabolism in rats, by increasing creatine kinase activity in prefrontal cortex, hippocampus and striatum, and increasing citrate synthase and succinate dehydrogenase activities in prefrontal cortex, hippocampus, striatum and cerebral cortex of rats (Reference Santos, Scaini and Rezin48,Reference Scaini, Santos and Benedet49).
Several studies showed that paroxetine, venlafaxine and nortriptyline produced anti-immobility effects in the forced swimming test, suggesting an antidepressant-like action in mice and rats (Reference Berrocoso, Rojas-Corrales and Micó32,Reference Ide, Fujiwara and Fujiwara35,Reference Consoni, Vital and Andreatini50,Reference Krass, Wegener, Vasar and Volke51). Interestingly, some evidence point out to the possibility that other drugs used for the treatment of mental disorders also modulate energy metabolism, including fluoxetine (Reference Gamaro, Streck, Matté, Prediger, Wyse and Dalmaz45). Furthermore, electroconvulsive shock, which is also used as therapy for depression, was shown to elicit energetic disturbance in rats, by decreasing Na+, K+-ATPase and creatine kinase activities (Reference Streck, Feier and Búrigo52,Reference Búrigo, Roza and Bassani53).
Paroxetine enhances serotonergic transmission by blocking the presynaptic active membrane transport mechanism for the reuptake of serotonin (Reference Johnson15,Reference Richelson16), nortriptyline blocks norepinephrine and serotonin uptake, and venlafaxine is an inhibitor of both serotonin and norepinephrine transport (Reference Richelson16,Reference Duman, Heninger and Nestler17). Taking together the present findings and other reports showing that fluoxetine (which also acts on the serotonergic synapse) modulate brain metabolism, we speculate whether alterations in serotonergic synapse caused by these drugs are related to the biochemical effects, especially the increase in several parameters of energy metabolism in the brain.
Most antidepressants need chronic administration before they achieve clinical effects; the mechanisms involved in this delay are not known, but their therapeutic efficacy is probably mediated by long-term molecular adaptations. In this context, several studies showed that some antidepressants, such as venlafaxine, alter the gene expression profile of human cells. For example, it has been reported that venlafaxine altered the expression of genes implicated in ionic homeostasis and genes associated with cell survival, neural plasticity, signal transduction and metabolism (Reference Kálmán, Palotás and Juhász54). On the other hand, nortriptyline modulated the expression of cytoskeleton proteins and carbohydrate metabolism, as well as proteins involved in rats and synaptic transmission and neurite morphogenesis pathways (Reference Piubelli, Gruber, El Khoury, Mathé, Domenici and Carboni55). Paroxetine also increased expression or modification of several proteins, including sepiapterin reductase, which controls the production of tetrahydrobiopterin, an essential cofactor for the synthesis of many neurotransmitters (Reference McHugh, Rogers, Loudon, Glubb, Joyce and Kennedy56).
In conclusion, we demonstrated that mitochondrial respiratory chain enzymes are activated in brain of adult rats after chronic administration of paroxetine, nortriptyline and venlafaxine. Considering that energy impairment may be involved in the pathophysiology of depressive disorders, we speculate that an increase in brain energy metabolism by antidepressant drugs could play a role in the mechanism of action of these drugs. These data corroborate with other studies suggesting that some antidepressants modulate brain energy metabolism.
Acknowledgements
This research was supported by grants from Programa de Pós-graduação em Ciências da Saúde – Universidade do Extremo Sul Catarinense (UNESC) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
