Cecilia Gabriel

Mechanisms Contributing to the Phase-Dependent Regulation of Neurogenesis by the Novel Antidepressant, Agomelatine, in the Adult Rat Hippocampus

Agomelatine is a novel antidepressant acting as a melatonergic receptor agonist and serotonergic (5-HT2C) receptor antagonist. In adult rats, chronic agomelatine treatment enhanced cell proliferation and neurogenesis in the ventral hippocampus (VH), a region pertinent to mood disorders. This study compared the effects of agomelatine on cell proliferation, maturation, and survival and investigated the cellular mechanisms underlying these effects. Agomelatine increased the ratio of mature vs immature neurons and enhanced neurite outgrowth of granular cells, suggesting an acceleration of maturation. The influence of agomelatine on maturation and survival was accompanied by a selective increase in the levels of BDNF (brain-derived neurotrophic factor) vs those of VEGF (vascular endothelial factor) and IGF-1 (insulin-like growth factor 1), which were not affected. Agomelatine also activated several cellular signals (extracellular signal-regulated kinase1/2, protein kinase B, and glycogen synthase kinase 3β) known to be modulated by antidepressants and implicated in the control of proliferation/survival. Furthermore, as agomelatine possesses both melatonergic agonist and serotonergic (5-HT2C) antagonist properties, we determined whether melatonin and 5-HT2C receptor antagonists similarly influence cell proliferation and survival. Only the 5-HT2C receptor antagonists, SB243,213 or S32006, but not melatonin, mimicked the effects of agomelatine on cell proliferation in VH. The promoting effect of agomelatine on survival was not reproduced by the 5-HT2C receptor antagonists or melatonin alone. However, it was blocked by a melatonin antagonist, S22153. These results show that agomelatine treatment facilitates all stages of neurogenesis and suggest that a joint effect of melatonin agonism and 5HT2C antagonism may be involved in promotion by agomelatine of survival in the hippocampus.

Behavioural and neuroplastic effects of the new-generation antidepressant agomelatine compared to fluoxetine in glucocorticoid receptor-impaired mice.

Major depression is associated with reduced hippocampal volume linked to stress and high glucocorticoid secretion. Glucocorticoid receptor-impaired (GR-i) mice, a transgenic model for affective disorders with hypothalamic-pituitary-adrenal (HPA) axis feedback control deficit, were used to assess the antidepressant-like effects of the mixed melatonin receptor agonist/5-HT(2C) receptor antagonist, agomelatine, compared to the selective 5-HT reuptake inhibitor (SSRI), fluoxetine, on hippocampal neurogenesis, GR and BDNF expression and antidepressant-responsive behaviour (tail suspension test, TST). GR-i and paired wild-type (WT) mice were given acute or chronic (21 d) treatment with these drugs. Both hippocampal cell proliferation and BDNF mRNA expression were down-regulated in GR-i mice, and these alterations were reversed by chronic agomelatine and fluoxetine treatments, whereas GR mRNA down-regulation was reversed only by agomelatine. Furthermore, chronic agomelatine, but not fluoxetine, increased survival of newly formed cells in the ventral part of the hippocampus without changing their phenotypic differentiation into neurons. In the TST, the enhanced immobility of GR-i mice was reduced to WT level by acute (but not chronic) fluoxetine and chronic (but not acute) agomelatine. These results indicate that agomelatine reversed the neuroplastic changes and helpless behaviour associated with HPA axis alterations in GR-i mice, suggesting neurobiological and behavioural effects mostly similar to those typically seen with classical antidepressants such as fluoxetine, but through clearly distinct mechanisms.

Beneficial behavioural and neurogenic effects of agomelatine in a model of depression/anxiety

Agomelatine (S20098) is a novel antidepressant drug with melatonergic agonist and 5-HT2C receptor antagonist properties, displaying antidepressant/anxiolytic-like properties in animal models and in humans. In a depression/anxiety-like mouse model in which the response of the HPA axis is blunted, we investigated whether agomelatine could reverse behavioural deficits related to depression/anxiety compared to the classical selective serotonin reuptake inhibitor, fluoxetine. Adult mice were treated for 8 wk with either vehicle or corticosterone (35 μg/ml.d) via drinking water. During the final 4 wk, animals were treated with vehicle, agomelatine (10 or 40 mg/kg i.p.) or fluoxetine (18 mg/kg i.p.) and tested in several behavioural paradigms and also evaluated for home-cage activity. Our results showed that the depressive/anxiety-like phenotype induced by corticosterone treatment is reversed by either chronic agomelatine or fluoxetine treatment. Moreover, agomelatine increased the dark/light ratio of home-cage activity in vehicle-treated mice and reversed the alterations in this ratio induced by chronic corticosterone, suggesting a normalization of disturbed circadian rhythms. Finally, we investigated the effects of this new antidepressant on neurogenesis. Agomelatine reversed the decreased cell proliferation in the whole hippocampus in corticosterone-treated mice and increased maturation of newborn neurons in both vehicle- and corticosterone-treated mice. Overall, the present study suggests that agomelatine, with its distinct mechanism of action based on the synergy between the melatonergic agonist and 5-HT2C antagonist properties, provides a distinct antidepressant/anxiolytic spectrum including circadian rhythm normalization.

Agomelatine reverses the decrease in hippocampal cell survival induced by chronic mild stress.

The antidepressant agomelatine is a MT(1)/MT(2) receptor agonist and 5-HT(2C) antagonist. Its antidepressant activity is proposed to result from the synergy between these sets of receptors. Agomelatine-induced changes in the brain have been reported under basal conditions. Yet, little is known about its effects in the brain exposed to chronic stress as a risk factor for major depressive disorder. Recently, we described agomelatine-induced changes on neuronal activity and adult neurogenesis in the hippocampus of rats subjected to chronic footshock stress. In order to better characterize the actions of agomelatine in the stress-compromised brain, here we investigated its effects on hippocampal neurogenesis in the chronic mild stress (CMS) model. Adult male rats were subjected to various mild stressors for 5 weeks, and treated with agomelatine during the last 3 weeks of the stress period. The sucrose preference test was performed weekly to measure anhedonia, and the marble burying test was carried out at the end of the experiment to assess anxiety-like behavior. In our model, the CMS paradigm did not change sucrose preference; however, it increased marble burying behavior, indicating enhanced anxiety. Interestingly, this stress model differentially affected distinct stages of the neurogenesis process. Whereas CMS did not influence the rate of hippocampal cell proliferation, it significantly decreased the newborn cell survival and doublecortin expression in the dentate gyrus. Importantly, treatment with agomelatine completely normalized stress-affected cell survival and partly reversed reduced doublecortin expression. Taken together, these data show that agomelatine has beneficial effects on hippocampal neurogenesis in the CMS paradigm.

Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry.

Mitochondrial membrane potential (MMP) in dissociated rat cerebellar neurons was measured using rhodamine 123 (Rh 123) as fluorescent dye, and flow cytometry. Dye distribution was studied by confocal scanning microscopy. Propidium iodide (PI)-marked cells (dead cells) were not stained by Rh 123, while the green fluorescence of living cells was restricted to mitochondria. Incubation of cells with different ionophores resulted in a maximal inhibition of Rh 123 fluorescence of 27.0 +/- 5.9% (valinomycin), 55.6 +/- 7.2% (ionomycin), and 37.3 +/- 5.1% (gramicidin). Ionophores decreased cell viability at high concentrations, measured as the number of propidium iodide-marked cells. Exposure of cell suspensions to the mitochondrial specific uncoupling agent CCCP caused a decrease in Rh 123 fluorescence (40 +/- 6.1%). Conversely, oxidative stress induced by H2O2 did not affect Rh 123 fluorescence. Impairment of glucose bioavailability reduced Rh 123 fluorescence. 2-Deoxy-D-glucose decreased the MMP with a maximal inhibition of 24.0 +/- 4.4%. Lack of glucose in the incubation medium also resulted in a decrease in MMP. Moreover, application of L-glutamate and N-methyl-D-aspartate (NMDA) (the excitatory amino acids) decreased Rh 123 uptake in a dose-dependent manner, which suggests that the measurement of MMP in dissociated cerebellar neurons by flow cytometry is a suitable method to detect the activity of drugs acting on glutamate receptors.

The antidepressant agomelatine blocks the adverse effects of stress on memory and enables spatial learning to rapidly increase neural cell adhesion molecule (NCAM) expression in the hippocampus of rats

Agomelatine, a novel antidepressant with established clinical efficacy, acts as a melatonin receptor agonist and 5-HT(2C) receptor antagonist. As stress is a significant risk factor in the development of depression, we sought to determine if chronic agomelatine treatment would block the stress-induced impairment of memory in rats trained in the radial-arm water maze (RAWM), a hippocampus-dependent spatial memory task. Moreover, since neural cell adhesion molecule (NCAM) is known to be critically involved in memory consolidation and synaptic plasticity, we evaluated the effects of agomelatine on NCAM, and polysialylated NCAM (PSA-NCAM) expression in rats given spatial memory training with or without predator stress. Adult male rats were pre-treated with agomelatine (10 mg/kg i.p., daily for 22 d), followed by a single day of RAWM training and memory testing. Rats were given 12 training trials and then they were placed either in their home cages (no stress) or near a cat (predator stress). Thirty minutes later the rats were given a memory test trial followed immediately by brain extraction. We found that: (1) agomelatine blocked the predator stress-induced impairment of spatial memory; (2) agomelatine-treated stressed, as well as non-stressed, rats exhibited a rapid training-induced increase in the expression of synaptic NCAM in the ventral hippocampus; and (3) agomelatine treatment blocked the water-maze training-induced decrease in PSA-NCAM levels in both stressed and non-stressed animals. This work provides novel observations which indicate that agomelatine blocks the adverse effects of stress on hippocampus-dependent memory and activates molecular mechanisms of memory storage in response to a learning experience.

Synergistic mechanisms in the modulation of the neurotrophin BDNF in the rat prefrontal cortex following acute agomelatine administration.

Abstract Objectives. The aim of this study was to investigate the acute modulation of the neurotrophin Brain-derived neurotrophic factor (BDNF) by the novel antidepressant agomelatine and the relative contribution of its melatonergic and serotonergic receptor components. Methods. BDNF mRNA levels were measured in rat hippocampus and prefrontal cortex after acute administration of agomelatine, melatonin or the 5-HT2C antagonist S32006. Results. BDNF expression was significantly increased 16 h after acute agomelatine administration, an effect that follows a specific temporal profile, is limited to the prefrontal cortex and it is due to changes of specific neurotrophin transcripts. Moreover, the acute up-regulation of BDNF mRNA levels appears to be the result of a synergistic effect between the melatonergic properties of agomelatine as MT1/MT2 agonist and its serotonergic 5-HT2C antagonism, since either melatonin or the 5-HT2C antagonist S32006 does not mimic the effects of agomelatine. Conclusions. These data provide evidence that acute agomelatine treatment modulates the expression of BDNF through a functional interaction between melatonergic MT1/MT2 and serotonergic 5-HT2C receptors, supporting the notion that intracellular events can be regulated via a synergistic activity of different neuromodulatory systems.

The Effects of Antidepressant Treatment in Prenatally Stressed Rats Support the Glutamatergic Hypothesis of Stress-Related Disorders

Abnormalities of synaptic transmission in the hippocampus represent an integral part of the altered programming triggered by early life stress, which enhances the vulnerability to stress-related disorders in the adult life. Rats exposed to prenatal restraint stress (PRS) develop enduring biochemical and behavioral changes characteristic of an anxious/depressive-like phenotype. Most neurochemical abnormalities in PRS rats are found in the ventral hippocampus, a region that encodes memories related to stress and emotions. We have recently demonstrated a causal link between the reduction of glutamate release in the ventral hippocampus and anxiety-like behavior in PRS rats. To confer pharmacological validity to the glutamatergic hypothesis of stress-related disorders, we examined whether chronic treatment with two antidepressants with different mechanisms of action could correct the defect in glutamate release and associated behavioral abnormalities in PRS rats. Adult unstressed or PRS rats were treated daily with either agomelatine (40 mg/kg, i.p.) or fluoxetine (5 mg/kg, i.p.) for 21 d. Both treatments reversed the reduction in depolarization-evoked glutamate release and in the expression of synaptic vesicle-associated proteins in the ventral hippocampus of PRS rats. Antidepressant treatment also corrected abnormalities in anxiety-/depression-like behavior and social memory performance in PRS rats. The effect on glutamate release was strongly correlated with the improvement of anxiety-like behavior and social memory. These data offer the pharmacological demonstration that glutamatergic hypofunction in the ventral hippocampus lies at the core of the pathological phenotype caused by early life stress and represents an attractive pharmacological target for novel therapeutic strategies.

The Novel Antidepressant Agomelatine Normalizes Hippocampal Neuronal Activity and Promotes Neurogenesis in Chronically Stressed Rats

Agomelatine is a novel antidepressant which acts as a melatonergic (MT1/MT2) receptor agonist and serotonergic (5‐HT2C) receptor antagonist. The antidepressant properties of agomelatine have been demonstrated in animal models as well as in clinical studies. Several preclinical studies reported agomelatine‐induced effects on brain plasticity, mainly under basal conditions in healthy animals. Yet, it is important to unravel agomelatine‐mediated changes in the brain affected by psychopathology or exposed to conditions that might predispose to mood disorders. Since stress is implicated in the etiology of depression, it is valid to investigate antidepressant‐induced effects in animals subjected to chronic stress. In this context, we sought to determine changes in the brain after agomelatine treatment in chronically stressed rats. Adult male rats were subjected to footshock stress and agomelatine treatment for 21 consecutive days. Rats exposed to footshock showed a robust increase in adrenocorticotropic hormone (ACTH) and corticosterone. Chronic agomelatine treatment did not markedly influence this HPA‐axis response. Whereas chronic exposure to daily footshock stress reduced c‐Fos expression in the hippocampal dentate gyrus, agomelatine treatment reversed this effect and normalized neuronal activity to basal levels. Moreover, chronic agomelatine administration was associated with enhanced hippocampal cell proliferation and survival in stressed but not in control rats. Furthermore, agomelatine reversed the stress‐induced decrease in doublecortin expression in the dentate gyrus. Taken together, these data show a beneficial action of agomelatine in the stress‐compromised brain, where it restores stress‐affected hippocampal neuronal activity and promotes adult hippocampal neurogenesis.

DETERMINATION OF NITRIC OXIDE GENERATION IN MAMMALIAN NEURONS USING DICHLOROFLUORESCIN DIACETATE AND FLOW CYTOMETRY

A method for the rapid detection of intracellular nitric oxide (NO) generation in dissociated cerebellar granule cells using dichlorofluorescin (DCFH) and flow cytometry was developed. DCFH can be oxidized specifically by NO and this was assessed by 1) the use of SIN-1 (10 nM-100 microM), an NO donor, that induced a concentration-dependent increase in dichlorofluorescein (DCF) fluorescence and 2) the use of hemoglobin (10 microM), an NO-scavenger, that totally inhibited the increase of fluorescence induced by SIN-1 (10 microM). This assay was used to determine the ability to kainate to stimulate NO production in dissociated cerebellar granule cells. Kainate (1 microM-10 mM) induced an increase in DCF fluorescence that was partially reduced by NG-nitro-L-arginine (1 nM-10 microM), a nitric oxide synthase inhibitor (61.9% +/- 9.1), or hemoglobin (10 microM) (55.0% +/- 4.1). The method described allows evaluation of the oxidation of DCFH to produce DCF as a parameter for measuring intracellular NO generation. The extent of DCFH oxidation by NO and ROS can be determined by using NO scavengers or NO synthase inhibitors.