BACKGROUND
despite the improved tolerability of newer antidepressants such as the selective serotonin reuptake inhibitors   <cit>  and the dual serotonin-norepinephrine reuptake inhibitors , adverse events associated with their use can influence adherence rates.
there is an unmet need for a better understanding of the mechanisms and therapeutic management of depression, as well as for treatments with improved efficacy and tolerability.
indeed, both the pathophysiology of depression and the exact mechanisms whereby antidepressants elicit a therapeutic response have not been fully explored.
an increasing body of evidence indicates that depression is associated with a disruption of circadian rhythms, suggesting that resetting of disrupted circadian rhythms may play a pivotal role in the treatment of this condition  <cit> .
agomelatine is a melatonergic receptor  agonist  <cit>  and 5ht2c receptor antagonist  <cit>  that has showed antidepressant efficacy in animal models  <cit>  and clinical trials  <cit> .
recent findings have shown that, in addition to the changes induced in monoamine transmission, antidepressants may work by modulating glutamate release and transmission in relevant limbic and cortical areas  <cit> .
in this regard, we have previously found that different chronic antidepressant treatments  abolish the increase of depolarization-evoked glutamate release, induced by acute footshock -stress, from synaptosomes of prefrontal/frontal cortex   <cit> .
here we studied the two separate components in the mechanism of agomelatine, by treating rats chronically with either agomelatine, or melatonin , or s <dig> , and then subjected the rats to fs-stress as above.
aim of the experiments was to investigate whether one of the two receptorial components was sufficient for the dampening action of agomelatine on stress-induced glutamate release, or both components were necessary.
we found that chronic treatment with either melatonin or s <dig> did not significantly reduce the stress-induced glutamate release, while chronic agomelatine treatment, as observed previously, completely abolished the stress-induced glutamate release, suggesting a potential synergy between mt1/mt2- and 5-ht2c-dependent pathways in the action of agomelatine.
RESULTS
depolarization-evoked release of glutamate from p/fc synaptosomes is increased after acute footshock stress
after chronic drug treatments, rats were subjected to standard acute fs-stress protocol  and, immediately after the stress session, the synaptosomes were purified from p/fc.
the presynaptic release of endogenous glutamate and gaba evoked by  <dig> mm kcl were measured from synaptosomes in superfusion, as done previously  <cit> .
one way anova showed significant difference among groups .
indeed, acute fs-stress increased depolarization-dependent release of endogenous glutamate from p/fc synaptosomes  and, as previously observed with several different antidepressants  <cit> , chronic agomelatine treatment completely prevented the stress-induced increase of glutamate release .
in the previous work  <cit>  we also observed that agomelatine, as well as other antidepressants, did not modify glutamate release from p/fc synaptosomes in non-stressed rats .
for this reason agomelatine treatment of non-stressed rats was not replicated here; similarly melatonin or s <dig> treatment of non-stressed rats was not performed here.
having agomelatine per se no effect on glutamate release in p/fc in the absence of stress, there was no point in testing if either of the two other compounds per se may modify glutamate release.
as shown in figure  <dig>  in rats treated with melatonin and then stressed, the release of glutamate was similarly increased as in stressed rats.
in rats treated with s <dig>  a trend for reduction of stress-induced glutamate release was observed  but the compound did not significantly dampen stress-induced glutamate release.
no significant changes in gaba release were induced by either fs-stress or any of the drugs.
no effect of acute agomelatine treatment  was found on fs-stress increased release of endogenous glutamate.
discussion and
CONCLUSIONS
in this study we sought to dissect the action of different receptorial components of agomelatine, with regard to the action of this drug on stress-induced glutamate release in p/fc.
this was carried out by performing chronic treatments with either melatonin or a selective 5-ht2c receptor antagonist , both endowed with receptorial affinity comparable with the affinity of agomelatine for the respective receptors.
the present results, showing that only agomelatine but not the two other compounds significantly prevented the stress-induced increase of glutamate release, suggest that the action of agomelatine on glutamate release requires both receptorial components.
however, it is worth noting that while glutamate release in melatonin-treated rats was virtually indistinguishable from stressed rats, in rats treated with s <dig> a trend for reduction of release was observed .
it may be speculated that enhancement of noradrenergic and dopaminergic transmission induced by 5-ht2c receptor antagonism  <cit>  in cortical areas is a necessary modification for dampening of glutamate release, similar to the outcome of treatments with monoamine reuptake inhibitors, which also increase monoamine neurotransmission with a different mechanism .
however, in the case of agomelatine only the combination of melatonergic agonism with 5-ht2c receptor antagonism seems to be sufficient for full blockade of stress-induced glutamate release.
this is in turn suggestive of a possible synergy between melatonin and 5-ht2c receptor-dependent pathways in the action of this drug.
these results are in line with the behavioral data published by papp et al.
<cit> , showing that the antidepressive effects of agomelatine in the chronic mild stress model of depression depend on a combination of its melatonin agonist and 5ht2c antagonist properties.
furthermore, comparable results were also obtained in the learned helplessness model of depression, where antidepressant effect was observed with agomelatine, but not after treatment with melatonin or the selective 5-ht2c antagonist sb <dig>  <cit> .
in this line, recently it has been demonstrated that the effects of chronic agomelatine on cell survival in hippocampus as well as on brain derived neurotrophic factor  expression maybe due to a synergy between both properties of the drug  <cit> .
further studies, at the level of intracellular signaling and of presynaptic machinery  <cit> , will investigate the suggested cross-talk between post-receptor pathways in the action of agomelatine on glutamate release.

 despite the improved tolerability of newer antidepressants such as the selective serotonin reuptake inhibitors and the dual serotonin-norepinephrine reuptake inhibitors, adverse events associated with their use can influence adherence rates. there is an unmet need for a better understanding of the mechanisms and therapeutic management of depression, as well as for treatments with improved efficacy and tolerability. indeed, both the pathophysiology of depression and the exact mechanisms whereby antidepressants elicit a therapeutic response have not been fully explored. an increasing body of evidence indicates that depression is associated with a disruption of circadian rhythms, suggesting that resetting of disrupted circadian rhythms may play a pivotal role in the treatment of this condition. agomelatine is a melatonergic receptor agonist and 5ht2c receptor antagonist that has showed antidepressant efficacy in animal models and clinical trials. recent findings have shown that, in addition to the changes induced in monoamine transmission, antidepressants may work by modulating glutamate release and transmission in relevant limbic and cortical areas. in this regard, we have previously found that different chronic antidepressant treatments abolish the increase of depolarization-evoked glutamate release, induced by acute footshock -stress, from synaptosomes of prefrontal/frontal cortex. here we studied the two separate components in the mechanism of agomelatine, by treating rats chronically with either agomelatine, or melatonin, or s, and then subjected the rats to fs-stress as above. aim of the experiments was to investigate whether one of the two receptorial components was sufficient for the dampening action of agomelatine on stress-induced glutamate release, or both components were necessary. we found that chronic treatment with either melatonin or s did not significantly reduce the stress-induced glutamate release, while chronic agomelatine treatment, as observed previously, completely abolished the stress-induced glutamate release, suggesting a potential synergy between mt1/mt2- and 5-ht2c-dependent pathways in the action of agomelatine.  depolarization-evoked release of glutamate from p/fc synaptosomes is increased after acute footshock stress after chronic drug treatments, rats were subjected to standard acute fs-stress protocol and, immediately after the stress session, the synaptosomes were purified from p/fc. the presynaptic release of endogenous glutamate and gaba evoked by mm kcl were measured from synaptosomes in superfusion, as done previously. one way anova showed significant difference among groups. indeed, acute fs-stress increased depolarization-dependent release of endogenous glutamate from p/fc synaptosomes and, as previously observed with several different antidepressants, chronic agomelatine treatment completely prevented the stress-induced increase of glutamate release. in the previous work we also observed that agomelatine, as well as other antidepressants, did not modify glutamate release from p/fc synaptosomes in non-stressed rats. for this reason agomelatine treatment of non-stressed rats was not replicated here; similarly melatonin or s treatment of non-stressed rats was not performed here. having agomelatine per se no effect on glutamate release in p/fc in the absence of stress, there was no point in testing if either of the two other compounds per se may modify glutamate release. as shown in figure in rats treated with melatonin and then stressed, the release of glutamate was similarly increased as in stressed rats. in rats treated with s a trend for reduction of stress-induced glutamate release was observed but the compound did not significantly dampen stress-induced glutamate release. no significant changes in gaba release were induced by either fs-stress or any of the drugs. no effect of acute agomelatine treatment was found on fs-stress increased release of endogenous glutamate. discussion and  in this study we sought to dissect the action of different receptorial components of agomelatine, with regard to the action of this drug on stress-induced glutamate release in p/fc. this was carried out by performing chronic treatments with either melatonin or a selective 5-ht2c receptor antagonist, both endowed with receptorial affinity comparable with the affinity of agomelatine for the respective receptors. the present results, showing that only agomelatine but not the two other compounds significantly prevented the stress-induced increase of glutamate release, suggest that the action of agomelatine on glutamate release requires both receptorial components. however, it is worth noting that while glutamate release in melatonin-treated rats was virtually indistinguishable from stressed rats, in rats treated with s a trend for reduction of release was observed. it may be speculated that enhancement of noradrenergic and dopaminergic transmission induced by 5-ht2c receptor antagonism in cortical areas is a necessary modification for dampening of glutamate release, similar to the outcome of treatments with monoamine reuptake inhibitors, which also increase monoamine neurotransmission with a different mechanism. however, in the case of agomelatine only the combination of melatonergic agonism with 5-ht2c receptor antagonism seems to be sufficient for full blockade of stress-induced glutamate release. this is in turn suggestive of a possible synergy between melatonin and 5-ht2c receptor-dependent pathways in the action of this drug. these results are in line with the behavioral data published by papp et al., showing that the antidepressive effects of agomelatine in the chronic mild stress model of depression depend on a combination of its melatonin agonist and 5ht2c antagonist properties. furthermore, comparable results were also obtained in the learned helplessness model of depression, where antidepressant effect was observed with agomelatine, but not after treatment with melatonin or the selective 5-ht2c antagonist sb. in this line, recently it has been demonstrated that the effects of chronic agomelatine on cell survival in hippocampus as well as on brain derived neurotrophic factor expression maybe due to a synergy between both properties of the drug. further studies, at the level of intracellular signaling and of presynaptic machinery, will investigate the suggested cross-talk between post-receptor pathways in the action of agomelatine on glutamate release. 
these results suggest a potential synergy between melatonergic and serotonergic pathways in the action of agomelatine. agomelatine is a melatonergic receptor agonist and a 5ht2c receptor antagonist that has shown antidepressant efficacy. only chronic agomelatine, but not melatonin or s completely prevented the stress-induced increase of glutamate release in the rat prefrontal/frontal cortex. in order to analyze separately the effect of the two receptorial components, rats were chronically treated with agomelatine, melatonin, or s, and then subjected to acute footshock-stress.
 agomelatine is a melatonergic receptor agonist and a 5ht2c receptor antagonist that has shown antidepressant efficacy. in order to analyze separately the effect of the two receptorial components, rats were chronically treated with agomelatine, melatonin, or s, and then subjected to acute footshock-stress.  only chronic agomelatine, but not melatonin or s completely prevented the stress-induced increase of glutamate release in the rat prefrontal/frontal cortex.  these results suggest a potential synergy between melatonergic and serotonergic pathways in the action of agomelatine. 