BACKGROUND
regulation of gene expression represents a major component in the mechanism of action of antidepressants  <cit> .
converging evidence shows that a common target of chronic antidepressants is a positive modulation of camp-responsive element binding protein , a transcription factor that regulates the expression of several genes involved in the control of neuroplasticity, circadian rhythms, cell survival and cognition  <cit> .
creb transcriptional activity is regulated by phosphorylation at ser <dig>  which is induced by multiple signalling cascades.
in particular, it was shown that the calcium/calmodulin -dependent and the mitogen activated protein  kinase cascades have a crucial role in the activation of creb consequent to chronic antidepressant treatments  <cit> .
moreover, recent preclinical biochemical and behavioural evidence suggested an involvement of the akt/glycogen synthase kinase- <dig>  signalling, regulating gene expression through the activation of creb and other transcription factors, both in the modulation of behaviour and in the mechanism of action of psychoactive drugs  <cit> .
gsk- <dig> is a ser/thr kinase regulated predominantly in an inhibitory manner through phosphorylation at n-terminal serine residues  by several protein kinases, including akt  <cit> .
gsk-3β has been linked to bipolar disorder, depression, and schizophrenia  <cit> .
it was recently shown that selective serotonin reuptake inhibitors  and other 5-ht-related antidepressants  inhibit gsk-3β in many brain regions  <cit>  and gsk- <dig> inhibitors showed antidepressant-like action in behavioural tests  <cit> .
gsk- <dig> was also reported to be required for the rapid antidepressant actions of ketamine  <cit> .
the antidepressant agomelatine is an agonist of melatonergic mt1/mt <dig> receptors and an antagonist of 5-ht2c receptors.
the unique and novel pharmacological profile of agomelatine has been found to be effective in the treatment of depressive symptoms, with a rapid stabilisation of circadian rhythms and a favourable tolerability profile.
efficacy of agomelatine in behavioral tests has been previously reported in different animal models  <cit> .
thus far, little is known about the signalling pathways affected by agomelatine downstream of receptor modulation.
this is especially interesting with regard to the short half-life  of agomelatine, its mechanism of action and the signalling pathways responsible for the changes in gene expression and therapeutic effect.
in this study, we verified whether agomelatine modulates the time-dependent activity of some major signalling pathways known to be regulated by traditional antidepressants.
in particular, the expression and activation  of creb, αcam kinase ii, map kinase/erk1/ <dig>  akt and gsk-3β were investigated at both nuclear and cytoplasmic level of hippocampus  and prefrontal and frontal cortex  of rats treated for three weeks with agomelatine or vehicle and sacrificed 1 h  or 16 h  after the last drug administration.
we found that agomelatine, rather than potentiating creb-related signalling as traditional antidepressants, was able to partly modulate the time-dependent activation of the map/erk1/ <dig> and akt/gsk cascades, suggesting that its antidepressant properties might be related with a fine-tuning of the time-dependent oscillations in forebrain intracellular signalling.
RESULTS
chronic agomelatine does not activate creb
in order to dissect the effect of chronic agomelatine on the signalling pathways regulating creb activity and its putative time-dependent modulation, rats were chronically treated with vehicle or agomelatine  at 5 p.m. and sacrificed after 1 h  or 16 hours .
creb expression and activation  were measured in pfc/fc and hpc nuclear fractions.
the two-way anova highlighted no time-related changes in creb expression or phosphorylation levels between 1 h or 16 h post-treatment and, interestingly, differently from traditional antidepressants  <cit> , chronic agomelatine did not induce any significant changes in creb expression or phosphorylation .figure  <dig>
time-dependent fluctuation of creb, αcam kinase ii, mapk/erk1/ <dig> in pfc/fc nuclei.
modulation by chronic agomelatine.
expression levels of creb , αcam kinase ii , mapk/erk <dig>  and mapk/erk <dig>  and phosphorylation levels of creb at ser133
, αcam kinase ii at thr286
, mapk/erk <dig> at thr202/204
and mapk/erk <dig> at thr202/204
in nuclear fraction of pfc/fc from rats chronically treated with vehicle  or agomelatine  and sacrificed  <dig> or 16 h after the last injection.
data are expressed as % intensity units/mm <dig> .
bonferroni post-hoc test : *p <  <dig>  veh treated rats sacrificed 16 h after last injection vs. veh treated rats sacrificed 1 h after last injection; § p <  <dig>  ago 16 h after last injection vs. ago 1 h after last injection; §§p <  <dig>  ago 16 h after last injection vs. ago 1 h after last injection .
time-dependent fluctuation of creb, αcam kinase ii, mapk/erk1/ <dig> in hpc nuclei.
modulation by chronic agomelatine.
expression levels of creb , αcam kinase ii , mapk/erk <dig>  and mapk/erk <dig>  and phosphorylation levels of creb at ser133
, αcam kinase ii at thr286
, mapk/erk <dig> at thr202/204
and mapk/erk <dig> at thr202/204
in nuclear fraction of hpc from rats chronically treated with vehicle  or agomelatine  and sacrificed  <dig> or 16 h after the last injection.
data expressed as above.
statistics as above.
**p <  <dig>  veh 16 h after last injection vs. veh 1 h after last injection; §§§p <  <dig>  ago 16 h after last injection vs. ago 1 h after last injection .
chronic agomelatine modulates time-dependent changes of mapk/erk1/ <dig> activation in frontal/prefrontal cortex but not in hippocampus
different intracellular signalling cascades, including cam kinases and map kinases, are known to modulate creb phosphorylation in the action of antidepressants  <cit> .
to investigate whether the lack of creb activation after chronic agomelatine was dependent on a lack of activation or a dysregulation of intracellular signalling cascades regulating creb phosphorylation, we assessed the expression and phosphorylation levels of αcam kinase ii and mapk/erk1/ <dig>  in pfc/fc and hpc nuclear fractions following agomelatine treatment.
interestingly, in both pfc/fc and hpc, we found that expression and phosphorylation levels of αcamkii were unmodified by both time and agomelatine .
on the contrary, the activation of mapk/erk1/ <dig>  through phosphorylation at thr202/tyr <dig> residues, was found to be significantly lower in animals sacrificed in the morning , than in the evening , in nuclei from both pfc/fc  =  <dig> , p <  <dig> ; perk2: f =  <dig> , p <  <dig> ) and hpc  =  <dig> , p <  <dig> ; perk2: f =  <dig> , p <  <dig> ) .
moreover, in pfc/fc, the 2-way anova highlighted a significant effect also of treatment for perk <dig>  =  <dig> , p <  <dig> ) , and the reduction of perk <dig> levels at  <dig> h was significant only for treated rats  , thus suggesting that agomelatine reduces erk <dig> activation and potentiates the time-dependent modulation of perk <dig> in pfc/fc.
on the contrary, the time-dependent changes in mapk/erk1/ <dig> phosphorylation were similar in hpc of treated and untreated animals .
modulation of time-dependent activation of the akt/gsk- <dig> pathway by chronic agomelatine in frontal/prefrontal cortex and hippocampus
to assess whether agomelatine can modulate the akt/gsk- <dig> signalling pathway, we measured total expression and phosphorylation levels of akt and gsk-3β in nuclear and cytosolic fractions from pfc/fc and hpc of rats chronically treated with agomelatine and sacrificed at  <dig> p.m. or  <dig> a.m. as described in details below, although the expression levels of akt and gsk-3β were unmodified by chronic agomelatine, the phosphorylation levels of the kinases were partly and selectively regulated by the drug, depending on the brain area and subcellular fraction analysed .
interestingly, in some cases, chronic agomelatine was able to modulate the activation of akt/gsk- <dig> phosphorylation, as shown below.figure  <dig>
time-dependent fluctuation of akt/gsk- <dig> signalling pathway in pfc/fc nuclear fraction.
modulation by chronic agomelatine.
expression levels of akt  and phosphorylation levels of akt at thr308
and at ser473
; expression levels of gsk- <dig> β ; phosphorylation levels of gsk- <dig> β at tyr216
and at ser9
in nuclear fraction of pfc/fc from rats chronically treated with vehicle  or agomelatine  and sacrificed  <dig> or 16 h after the last injection.
data expressed as above.
statistics as above.
**p <  <dig> , ***p <  <dig>  veh 16 h after last injection vs. veh 1 h after last injection; §§p < <dig>  ago 16 h after last injection vs. ago 1 h after last injection .
time-dependent fluctuation of akt/gsk- <dig> signalling pathway in pfc/fc cytosolic fraction and modulation by chronic agomelatine.
expression levels of akt  and phosphorylation levels of akt at thr308
and at ser473
; expression levels of gsk- <dig> β ; phosphorylation levels of gsk- <dig> β at tyr216
and at ser9
in cytosolic fraction of pfc/fc from rats chronically treated with vehicle  or agomelatine  and sacrificed  <dig> or 16 h after the last injection.
data expressed as above.
statistics as above.
*p <  <dig>  veh 16 h after last injection vs. veh 1 h after last injection; §p <  <dig>  ago 16 h after last injection vs. ago 1 h after last injection .
time-dependent fluctuation of akt/gsk- <dig> signalling pathway in hpc nuclear fraction and modulation by chronic agomelatine.
expression levels of akt  and phosphorylation levels of akt at thr308
and at ser473
; expression levels of gsk- <dig> β ; phosphorylation levels of gsk- <dig> β at tyr216
and at ser9
in nuclear fraction of hpc from rats chronically treated with vehicle  or agomelatine  and sacrificed  <dig> or 16 h after the last injection.
data expressed as above.
statistics as above.
**p <  <dig>  veh 16 h after last injection vs. veh 1 h after last injection; §p <  <dig> , §§p <  <dig>  ago 16 h after last injection vs. ago 1 h after last injection .
time-dependent fluctuation of akt/gsk- <dig> signalling pathway in hpc cytosolic fraction and modulation by chronic agomelatine.
expression levels of akt  and phosphorylation levels of akt at thr308
and at ser473
; expression levels of gsk- <dig> β ; phosphorylation levels of gsk- <dig> β at tyr216
and at ser9
in cytosolic fraction of hpc from rats chronically treated with vehicle  or agomelatine  and sacrificed  <dig> or 16 h after the last injection.
data expressed as above.
statistics as above.
*p <  <dig>  veh 16 h after last injection vs. veh 1 h after last injection; §§p <  <dig>  ago 16h after last injection vs. ago 1h after last injection .
in nuclear fraction from pfc/fc, the 2-way anova showed a significant effect of time on akt phosphorylation at thr <dig>  which is decreased in rats sacrificed in the morning  =  <dig>  p <  <dig> ) .
interestingly, post-hoc test was significant only for vehicle treated rats  , suggesting that agomelatine dampens the time-dependent modulation of phospho-thr <dig> akt.
instead, 2-way anova reported a significant effect of both time and treatment for phospho-ser <dig> akt, whose levels were decreased by agomelatine and were significantly lower in both groups of animals at 16 h vs 1 h  =  <dig>  p <  <dig> , treatment f =  <dig>  p <  <dig> ; bonferroni post-hoc test: veh 16 h vs. 1 h − <dig> % p <  <dig> , ago 16 h vs. 1 h − <dig> % p <  <dig> ) .
a significant effect of time was also found in nuclear fraction from pfc/fc for phospho-ser <dig> gsk-3β, which levels were lower in both agomelatine and vehicle treated rats sacrificed in the morning  =  <dig>  p <  <dig> ; bonferroni post-hoc tests: veh 16 h vs. 1 h − <dig> % p <  <dig> , ago 16 h vs. 1 h − <dig> % p <  <dig> ) .
in cytosol from pfc/fc, a significant effect of time was found on phospho-ser <dig> akt levels  =  <dig>  p <  <dig> ), while post-hoc comparisons were significant only in vehicle treated rats  .
moreover, 2-way anova showed a significant effect of time on phosphorylation of gsk-3β at tyr <dig>  =  <dig>  p <  <dig> ) and the post-hoc test highlighted a significant increase at 16 h selectively in rats chronically treated with agomelatine  .
no significant differences were found in the phosphorylation state of both ptyr <dig> of akt and pser <dig> of gsk-3β in cytosol from pfc/fc .
a different pattern was found in hpc.
indeed, in the nuclear fraction from hpc, while phospho-thr <dig> akt levels were similarly reduced in the morning in all animals  =  <dig>  p <  <dig> ; bonferroni post-hoc tests: veh 16 h vs. 1 h − <dig> % p <  <dig> , ago 16 h vs. 1 h − <dig> % p <  <dig> ) , the time-dependent reduction of phosphorylation of akt at ser <dig> was significant only in rats treated with agomelatine  =  <dig>  p <  <dig> ; bonferroni post-hoc test: ago 16 h vs. 1 h − <dig> % p <  <dig> ) .
no differences were found in the phosphorylation state of both ptyr <dig> and ser <dig> of gsk-3β in hpc nuclear fraction .
in the cytosolic fraction from the same area, the 2-way anova highlighted a significant effect of time for phospho-thr <dig> akt levels  =  <dig>  p <  <dig> )  and of both time and treatment/time interaction for phospho-ser <dig> akt  =  <dig>  p <  <dig> ; treatment/time interaction f =  <dig>  p <  <dig> ) , thus suggesting that agomelatine exerts a different effect at 16 h vs 1 h. post-hoc test showed a significant morning decrease of phospho-thr <dig> akt levels only in vehicle, but not in agomelatine treated rats   and a significant and marked reduction of phospho-ser <dig> akt levels at 16 h  .
no difference was found in gsk-3β phosphorylation at both tyr <dig> and ser <dig> residues in hpc cytosol .
for clarity, all significant changes in time-dependent regulation of mapk/erk1/ <dig> and akt/gsk- <dig> pathways, in nuclei and cytosol from pfc/fc and hpc of vehicle or agomelatine treated rats, and relative statistics are resumed in table  <dig> table  <dig>
summarizing significant changes in time-dependent regulation of mapk/erk1/ <dig> and akt/gsk- <dig> pathways, in nuclei and cytosol from pfc/fc and hpc of vehicle  or agomelatine  treated rats, sacrificed 1 h or 16 h after last injection
protein
brain area
subcellular fraction
variable
f statistic
<dig> way anova p value
significant bonferroni post-hoc tests
time
f =  <dig>
p <  <dig>
treatment
f =  <dig>
p <  <dig>
time
f =  <dig>
p <  <dig>
time
f =  <dig>
p <  <dig>
ago 1 h p <  <dig>
time
f =  <dig>
p <  <dig>
ago 1 h p <  <dig>
time
f =  <dig>
p <  <dig>
treatment
f =  <dig>
p <  <dig>
time
f =  <dig>
p <  <dig>
ago 1 h p <  <dig>
time
f =  <dig>
p <  <dig>
ago 1 h p <  <dig>
time
f =  <dig>
p <  <dig>
time
f =  <dig>
p <  <dig>
time
f =  <dig>
p <  <dig>
ago 1 h p <  <dig>
time
f =  <dig>
p <  <dig>
time
f =  <dig>
p <  <dig>
time
f =  <dig>
p <  <dig>
time
f =  <dig>
p <  <dig>
interaction
f =  <dig>
p <  <dig>
significant effects are highlighted in bold.
discussion
the study of the effects of agomelatine on intracellular signalling pathways modulating creb activation demonstrated that, differently from other antidepressants that activate the transcription factor creb, mainly through phosphorylation by αcam kinase ii and iv and mapk/erk1/ <dig> cascades  <cit> , chronic agomelatine did not act on these molecular mechanisms.
indeed, we did not observe any significant modification in both creb and αcam kinase ii activation in nuclear fraction from pfc/fc or hpc.
our results are in line with those of morley-fletcher et al.
<cit> , who recently showed in the same rat strain, that chronic agomelatine, although able to reverse the reduction of creb phosphorylation induced by prenatal stress in rats, had no effect on creb phosphorylation in non-stressed animals.
a number of previous studies have shown that both acute and chronic agomelatine increase the expression of brain-derived neurotrophic factor and of other neuroplastic molecules such as fibroblast growth factor, and activity-regulated cytoskeleton-associated protein  <cit> , suggesting that agomelatine could exert neurotrophic and antidepressant effects through the activation of molecular mechanisms partly different from those of other drugs  <cit> .
interestingly, we observed time-dependent changes in the activation of mapk/erk1/ <dig> kinases in both pfc/fc and hpc.
indeed, we found significantly lower phosphorylation levels of both kinases in animals sacrificed in the morning as compared to those sacrificed in the evening.
a circadian modulation of mapk/erk1/ <dig> activity was recently demonstrated in hippocampus and shown to contribute to memory consolidation  <cit> .
thus, according to our present results, time-related fluctuations in nuclear erk1/ <dig> activation are present also in pfc/fc, where chronic treatment with agomelatine seems to partly modulate the physiological changes.
further studies are needed to better clarify the circadian nature of these modifications and the potential of agomelatine in the modulation of the mapk/erk1/ <dig> signalling pathways.
we also observed a time-dependent modulation of akt and gsk-3β activation through phosphorylation that was selectively different depending on the brain area and the subcellular fraction analysed.
in particular, we found that akt phosphorylation was lower in pfc/fc and hpc of animals sacrificed in the morning .
this down-regulation of the kinase activation is particularly evident in the nuclear fraction, suggesting a possible involvement in the regulation of gene transcription.
moreover, since the ser <dig> of gsk-3β is phosphorylated by akt, the reduced phosphorylation of gsk-3β at ser <dig> observed in nuclei from pfc/fc could be linked to the morning decrease of akt activity observed in the same fraction.
although previous studies have shown a circadian modulation of akt and gsk- <dig> activity, the functional role of these fluctuations is poorly understood.
genetic manipulations, leading to increased akt activity in neurons of the suprachiasmatic nucleus, lengthen the circadian period of locomotor activity, whereas the reduction of akt signalling shortens it  <cit> .
moreover, it was recently demonstrated that both genetic and pharmacological reduction of gsk- <dig> activity in mouse embryonic fibroblasts have a specific effect on the circadian transcriptional oscillation, showing a phase delay in the transcription of the clock gene mper <dig>  <cit> .
our data suggest that chronic treatment with agomelatine reduces phospho-akt at ser <dig> in nuclear fraction of pfc/fc.
more interestingly, chronic agomelatine was also shown to partly modulate the time-dependent regulation of akt and gsk-3β activity.
indeed, agomelatine treatment dampened the morning reduction of akt phosphorylation at thr <dig> in nuclear fraction and at ser <dig> in cytosol of pfc/fc.
moreover, agomelatine induced a significant increase in ptyr <dig> gsk-3β in cytosol from pfc/fc and a decrease of pser <dig> akt in nuclei from hpc 16 h after the last drug injection.
finally, in the cytosolic fraction from hpc of rats sacrificed in the morning, agomelatine dampened the phosphorylation levels of akt at thr <dig> and induced a significant decrease of pser <dig> akt.
agomelatine treatment for  <dig> days was previously described to induce an increase in erk1/ <dig>  akt and gsk-3β phosphorylation after chronic treatment in whole hippocampal extract from hpc of rats sacrificed 16h after last drug administration  <cit> .
the following factors might contribute to explaining the apparent contrasting findings compared to our present results: 1- different rat strains were used, sprague dawley rats in our study vs. wistar rats in the previous study; 2- different antibodies were used; 3- the cellular fractions analysed in the two studies were different .
in this regard, it is well known that the subcellular distribution and compartmentalization of erk-mapks between cytosol and nucleus play a key role in the regulation of activity and specificity of the kinases  <cit> .
CONCLUSIONS
in summary, we have shown that chronic treatment with the antidepressant agomelatine does not activate creb and creb-related signalling in nuclei and cytosol from both hpc and pfc/fc of naïve rats.
this is of particular interest because suggests that the antidepressant effect of agomelatine could be mediated by different molecular mechanisms with respect to classical antidepressants.
although further studies are warranted to identify the different pattern of intracellular signalling pathways modulated by agomelatine, this difference might contribute to the therapeutic benefits.
moreover, although further studies are required to reveal whether mapk/erk1/ <dig> and akt/gsk- <dig> signalling pathways could play a role in the circadian control in hpc and pfc/fc, the present work has provided clear evidence of a time-dependent modulation of these pathways in both brain areas.
finally, considering the effects of agomelatine on the time-dependent regulation of erk1/ <dig>  akt and gsk-3β phosphorylation levels, it can be speculated that this could be a target in the antidepressant effect induced by the drug.

 regulation of gene expression represents a major component in the mechanism of action of antidepressants. converging evidence shows that a common target of chronic antidepressants is a positive modulation of camp-responsive element binding protein, a transcription factor that regulates the expression of several genes involved in the control of neuroplasticity, circadian rhythms, cell survival and cognition. creb transcriptional activity is regulated by phosphorylation at ser which is induced by multiple signalling cascades. in particular, it was shown that the calcium/calmodulin -dependent and the mitogen activated protein kinase cascades have a crucial role in the activation of creb consequent to chronic antidepressant treatments. moreover, recent preclinical biochemical and behavioural evidence suggested an involvement of the akt/glycogen synthase kinase- signalling, regulating gene expression through the activation of creb and other transcription factors, both in the modulation of behaviour and in the mechanism of action of psychoactive drugs. gsk- is a ser/thr kinase regulated predominantly in an inhibitory manner through phosphorylation at n-terminal serine residues by several protein kinases, including akt. gsk-3β has been linked to bipolar disorder, depression, and schizophrenia. it was recently shown that selective serotonin reuptake inhibitors and other 5-ht-related antidepressants inhibit gsk-3β in many brain regions and gsk- inhibitors showed antidepressant-like action in behavioural tests. gsk- was also reported to be required for the rapid antidepressant actions of ketamine. the antidepressant agomelatine is an agonist of melatonergic mt1/mt receptors and an antagonist of 5-ht2c receptors. the unique and novel pharmacological profile of agomelatine has been found to be effective in the treatment of depressive symptoms, with a rapid stabilisation of circadian rhythms and a favourable tolerability profile. efficacy of agomelatine in behavioral tests has been previously reported in different animal models. thus far, little is known about the signalling pathways affected by agomelatine downstream of receptor modulation. this is especially interesting with regard to the short half-life of agomelatine, its mechanism of action and the signalling pathways responsible for the changes in gene expression and therapeutic effect. in this study, we verified whether agomelatine modulates the time-dependent activity of some major signalling pathways known to be regulated by traditional antidepressants. in particular, the expression and activation of creb, αcam kinase ii, map kinase/erk1/ akt and gsk-3β were investigated at both nuclear and cytoplasmic level of hippocampus and prefrontal and frontal cortex of rats treated for three weeks with agomelatine or vehicle and sacrificed 1 h or 16 h after the last drug administration. we found that agomelatine, rather than potentiating creb-related signalling as traditional antidepressants, was able to partly modulate the time-dependent activation of the map/erk1/ and akt/gsk cascades, suggesting that its antidepressant properties might be related with a fine-tuning of the time-dependent oscillations in forebrain intracellular signalling.  chronic agomelatine does not activate creb in order to dissect the effect of chronic agomelatine on the signalling pathways regulating creb activity and its putative time-dependent modulation, rats were chronically treated with vehicle or agomelatine at 5 p.m. and sacrificed after 1 h or 16 hours. creb expression and activation were measured in pfc/fc and hpc nuclear fractions. the two-way anova highlighted no time-related changes in creb expression or phosphorylation levels between 1 h or 16 h post-treatment and, interestingly, differently from traditional antidepressants, chronic agomelatine did not induce any significant changes in creb expression or phosphorylation.figure time-dependent fluctuation of creb, αcam kinase ii, mapk/erk1/ in pfc/fc nuclei. modulation by chronic agomelatine. expression levels of creb, αcam kinase ii, mapk/erk and mapk/erk and phosphorylation levels of creb at ser133, αcam kinase ii at thr286, mapk/erk at thr202/204 and mapk/erk at thr202/204 in nuclear fraction of pfc/fc from rats chronically treated with vehicle or agomelatine and sacrificed or 16 h after the last injection. data are expressed as % intensity units/mm. bonferroni post-hoc test: *p <  veh treated rats sacrificed 16 h after last injection vs. veh treated rats sacrificed 1 h after last injection; § p <  ago 16 h after last injection vs. ago 1 h after last injection; §§p <  ago 16 h after last injection vs. ago 1 h after last injection. time-dependent fluctuation of creb, αcam kinase ii, mapk/erk1/ in hpc nuclei. modulation by chronic agomelatine. expression levels of creb, αcam kinase ii, mapk/erk and mapk/erk and phosphorylation levels of creb at ser133, αcam kinase ii at thr286, mapk/erk at thr202/204 and mapk/erk at thr202/204 in nuclear fraction of hpc from rats chronically treated with vehicle or agomelatine and sacrificed or 16 h after the last injection. data expressed as above. statistics as above. **p <  veh 16 h after last injection vs. veh 1 h after last injection; §§§p <  ago 16 h after last injection vs. ago 1 h after last injection. chronic agomelatine modulates time-dependent changes of mapk/erk1/ activation in frontal/prefrontal cortex but not in hippocampus different intracellular signalling cascades, including cam kinases and map kinases, are known to modulate creb phosphorylation in the action of antidepressants. to investigate whether the lack of creb activation after chronic agomelatine was dependent on a lack of activation or a dysregulation of intracellular signalling cascades regulating creb phosphorylation, we assessed the expression and phosphorylation levels of αcam kinase ii and mapk/erk1/ in pfc/fc and hpc nuclear fractions following agomelatine treatment. interestingly, in both pfc/fc and hpc, we found that expression and phosphorylation levels of αcamkii were unmodified by both time and agomelatine. on the contrary, the activation of mapk/erk1/ through phosphorylation at thr202/tyr residues, was found to be significantly lower in animals sacrificed in the morning, than in the evening, in nuclei from both pfc/fc  = , p < ; perk2: f = , p <  ) and hpc  = , p < ; perk2: f = , p <  ). moreover, in pfc/fc, the 2-way anova highlighted a significant effect also of treatment for perk  = , p <  ), and the reduction of perk levels at h was significant only for treated rats, thus suggesting that agomelatine reduces erk activation and potentiates the time-dependent modulation of perk in pfc/fc. on the contrary, the time-dependent changes in mapk/erk1/ phosphorylation were similar in hpc of treated and untreated animals. modulation of time-dependent activation of the akt/gsk- pathway by chronic agomelatine in frontal/prefrontal cortex and hippocampus to assess whether agomelatine can modulate the akt/gsk- signalling pathway, we measured total expression and phosphorylation levels of akt and gsk-3β in nuclear and cytosolic fractions from pfc/fc and hpc of rats chronically treated with agomelatine and sacrificed at p.m. or a.m. as described in details below, although the expression levels of akt and gsk-3β were unmodified by chronic agomelatine, the phosphorylation levels of the kinases were partly and selectively regulated by the drug, depending on the brain area and subcellular fraction analysed. interestingly, in some cases, chronic agomelatine was able to modulate the activation of akt/gsk- phosphorylation, as shown below.figure time-dependent fluctuation of akt/gsk- signalling pathway in pfc/fc nuclear fraction. modulation by chronic agomelatine. expression levels of akt and phosphorylation levels of akt at thr308 and at ser473; expression levels of gsk- β; phosphorylation levels of gsk- β at tyr216 and at ser9 in nuclear fraction of pfc/fc from rats chronically treated with vehicle or agomelatine and sacrificed or 16 h after the last injection. data expressed as above. statistics as above. **p < , ***p <  veh 16 h after last injection vs. veh 1 h after last injection; §§p < ago 16 h after last injection vs. ago 1 h after last injection. time-dependent fluctuation of akt/gsk- signalling pathway in pfc/fc cytosolic fraction and modulation by chronic agomelatine. expression levels of akt and phosphorylation levels of akt at thr308 and at ser473; expression levels of gsk- β; phosphorylation levels of gsk- β at tyr216 and at ser9 in cytosolic fraction of pfc/fc from rats chronically treated with vehicle or agomelatine and sacrificed or 16 h after the last injection. data expressed as above. statistics as above. *p <  veh 16 h after last injection vs. veh 1 h after last injection; §p <  ago 16 h after last injection vs. ago 1 h after last injection. time-dependent fluctuation of akt/gsk- signalling pathway in hpc nuclear fraction and modulation by chronic agomelatine. expression levels of akt and phosphorylation levels of akt at thr308 and at ser473; expression levels of gsk- β; phosphorylation levels of gsk- β at tyr216 and at ser9 in nuclear fraction of hpc from rats chronically treated with vehicle or agomelatine and sacrificed or 16 h after the last injection. data expressed as above. statistics as above. **p <  veh 16 h after last injection vs. veh 1 h after last injection; §p < , §§p <  ago 16 h after last injection vs. ago 1 h after last injection. time-dependent fluctuation of akt/gsk- signalling pathway in hpc cytosolic fraction and modulation by chronic agomelatine. expression levels of akt and phosphorylation levels of akt at thr308 and at ser473; expression levels of gsk- β; phosphorylation levels of gsk- β at tyr216 and at ser9 in cytosolic fraction of hpc from rats chronically treated with vehicle or agomelatine and sacrificed or 16 h after the last injection. data expressed as above. statistics as above. *p <  veh 16 h after last injection vs. veh 1 h after last injection; §§p <  ago 16h after last injection vs. ago 1h after last injection. in nuclear fraction from pfc/fc, the 2-way anova showed a significant effect of time on akt phosphorylation at thr which is decreased in rats sacrificed in the morning  =  p <  ). interestingly, post-hoc test was significant only for vehicle treated rats, suggesting that agomelatine dampens the time-dependent modulation of phospho-thr akt. instead, 2-way anova reported a significant effect of both time and treatment for phospho-ser akt, whose levels were decreased by agomelatine and were significantly lower in both groups of animals at 16 h vs 1 h  =  p < , treatment f =  p < ; bonferroni post-hoc test: veh 16 h vs. 1 h − % p < , ago 16 h vs. 1 h − % p <  ). a significant effect of time was also found in nuclear fraction from pfc/fc for phospho-ser gsk-3β, which levels were lower in both agomelatine and vehicle treated rats sacrificed in the morning  =  p < ; bonferroni post-hoc tests: veh 16 h vs. 1 h − % p < , ago 16 h vs. 1 h − % p <  ). in cytosol from pfc/fc, a significant effect of time was found on phospho-ser akt levels  =  p <  ), while post-hoc comparisons were significant only in vehicle treated rats. moreover, 2-way anova showed a significant effect of time on phosphorylation of gsk-3β at tyr  =  p <  ) and the post-hoc test highlighted a significant increase at 16 h selectively in rats chronically treated with agomelatine. no significant differences were found in the phosphorylation state of both ptyr of akt and pser of gsk-3β in cytosol from pfc/fc. a different pattern was found in hpc. indeed, in the nuclear fraction from hpc, while phospho-thr akt levels were similarly reduced in the morning in all animals  =  p < ; bonferroni post-hoc tests: veh 16 h vs. 1 h − % p < , ago 16 h vs. 1 h − % p <  ), the time-dependent reduction of phosphorylation of akt at ser was significant only in rats treated with agomelatine  =  p < ; bonferroni post-hoc test: ago 16 h vs. 1 h − % p <  ). no differences were found in the phosphorylation state of both ptyr and ser of gsk-3β in hpc nuclear fraction. in the cytosolic fraction from the same area, the 2-way anova highlighted a significant effect of time for phospho-thr akt levels  =  p <  ) and of both time and treatment/time interaction for phospho-ser akt  =  p < ; treatment/time interaction f =  p <  ), thus suggesting that agomelatine exerts a different effect at 16 h vs 1 h. post-hoc test showed a significant morning decrease of phospho-thr akt levels only in vehicle, but not in agomelatine treated rats and a significant and marked reduction of phospho-ser akt levels at 16 h. no difference was found in gsk-3β phosphorylation at both tyr and ser residues in hpc cytosol. for clarity, all significant changes in time-dependent regulation of mapk/erk1/ and akt/gsk- pathways, in nuclei and cytosol from pfc/fc and hpc of vehicle or agomelatine treated rats, and relative statistics are resumed in table  table summarizing significant changes in time-dependent regulation of mapk/erk1/ and akt/gsk- pathways, in nuclei and cytosol from pfc/fc and hpc of vehicle or agomelatine treated rats, sacrificed 1 h or 16 h after last injection protein brain area subcellular fraction variable f statistic way anova p value significant bonferroni post-hoc tests time f =  p <  treatment f =  p <  time f =  p <  time f =  p <  ago 1 h p <  time f =  p <  ago 1 h p <  time f =  p <  treatment f =  p <  time f =  p <  ago 1 h p <  time f =  p <  ago 1 h p <  time f =  p <  time f =  p <  time f =  p <  ago 1 h p <  time f =  p <  time f =  p <  time f =  p <  time f =  p <  interaction f =  p <  significant effects are highlighted in bold. discussion the study of the effects of agomelatine on intracellular signalling pathways modulating creb activation demonstrated that, differently from other antidepressants that activate the transcription factor creb, mainly through phosphorylation by αcam kinase ii and iv and mapk/erk1/ cascades, chronic agomelatine did not act on these molecular mechanisms. indeed, we did not observe any significant modification in both creb and αcam kinase ii activation in nuclear fraction from pfc/fc or hpc. our results are in line with those of morley-fletcher et al., who recently showed in the same rat strain, that chronic agomelatine, although able to reverse the reduction of creb phosphorylation induced by prenatal stress in rats, had no effect on creb phosphorylation in non-stressed animals. a number of previous studies have shown that both acute and chronic agomelatine increase the expression of brain-derived neurotrophic factor and of other neuroplastic molecules such as fibroblast growth factor, and activity-regulated cytoskeleton-associated protein, suggesting that agomelatine could exert neurotrophic and antidepressant effects through the activation of molecular mechanisms partly different from those of other drugs. interestingly, we observed time-dependent changes in the activation of mapk/erk1/ kinases in both pfc/fc and hpc. indeed, we found significantly lower phosphorylation levels of both kinases in animals sacrificed in the morning as compared to those sacrificed in the evening. a circadian modulation of mapk/erk1/ activity was recently demonstrated in hippocampus and shown to contribute to memory consolidation. thus, according to our present results, time-related fluctuations in nuclear erk1/ activation are present also in pfc/fc, where chronic treatment with agomelatine seems to partly modulate the physiological changes. further studies are needed to better clarify the circadian nature of these modifications and the potential of agomelatine in the modulation of the mapk/erk1/ signalling pathways. we also observed a time-dependent modulation of akt and gsk-3β activation through phosphorylation that was selectively different depending on the brain area and the subcellular fraction analysed. in particular, we found that akt phosphorylation was lower in pfc/fc and hpc of animals sacrificed in the morning. this down-regulation of the kinase activation is particularly evident in the nuclear fraction, suggesting a possible involvement in the regulation of gene transcription. moreover, since the ser of gsk-3β is phosphorylated by akt, the reduced phosphorylation of gsk-3β at ser observed in nuclei from pfc/fc could be linked to the morning decrease of akt activity observed in the same fraction. although previous studies have shown a circadian modulation of akt and gsk- activity, the functional role of these fluctuations is poorly understood. genetic manipulations, leading to increased akt activity in neurons of the suprachiasmatic nucleus, lengthen the circadian period of locomotor activity, whereas the reduction of akt signalling shortens it. moreover, it was recently demonstrated that both genetic and pharmacological reduction of gsk- activity in mouse embryonic fibroblasts have a specific effect on the circadian transcriptional oscillation, showing a phase delay in the transcription of the clock gene mper. our data suggest that chronic treatment with agomelatine reduces phospho-akt at ser in nuclear fraction of pfc/fc. more interestingly, chronic agomelatine was also shown to partly modulate the time-dependent regulation of akt and gsk-3β activity. indeed, agomelatine treatment dampened the morning reduction of akt phosphorylation at thr in nuclear fraction and at ser in cytosol of pfc/fc. moreover, agomelatine induced a significant increase in ptyr gsk-3β in cytosol from pfc/fc and a decrease of pser akt in nuclei from hpc 16 h after the last drug injection. finally, in the cytosolic fraction from hpc of rats sacrificed in the morning, agomelatine dampened the phosphorylation levels of akt at thr and induced a significant decrease of pser akt. agomelatine treatment for days was previously described to induce an increase in erk1/ akt and gsk-3β phosphorylation after chronic treatment in whole hippocampal extract from hpc of rats sacrificed 16h after last drug administration. the following factors might contribute to explaining the apparent contrasting findings compared to our present results: 1- different rat strains were used, sprague dawley rats in our study vs. wistar rats in the previous study; 2- different antibodies were used; 3- the cellular fractions analysed in the two studies were different. in this regard, it is well known that the subcellular distribution and compartmentalization of erk-mapks between cytosol and nucleus play a key role in the regulation of activity and specificity of the kinases.  in summary, we have shown that chronic treatment with the antidepressant agomelatine does not activate creb and creb-related signalling in nuclei and cytosol from both hpc and pfc/fc of naïve rats. this is of particular interest because suggests that the antidepressant effect of agomelatine could be mediated by different molecular mechanisms with respect to classical antidepressants. although further studies are warranted to identify the different pattern of intracellular signalling pathways modulated by agomelatine, this difference might contribute to the therapeutic benefits. moreover, although further studies are required to reveal whether mapk/erk1/ and akt/gsk- signalling pathways could play a role in the circadian control in hpc and pfc/fc, the present work has provided clear evidence of a time-dependent modulation of these pathways in both brain areas. finally, considering the effects of agomelatine on the time-dependent regulation of erk1/ akt and gsk-3β phosphorylation levels, it can be speculated that this could be a target in the antidepressant effect induced by the drug. 
we have found that in naïve rats chronic agomelatine, contrary to traditional antidepressants, did not increase creb phosphorylation, but modulates the time-dependent regulation of mapk/erk1/ and akt/glycogen synthase kinase- pathways. our results suggest that the intracellular molecular mechanisms modulated by chronic agomelatine may be partly different from those of traditional antidepressants and involve the time-dependent regulation of mapk/erk1/ and akt/gsk- signalling pathways. antidepressantagomelatineintracellular signallingneuroplasticitytime-dependent regulationissue-copyright-statement© the author 2014 aim of this work was to analyse possible effects of chronic agomelatine on time-dependent changes of different intracellular signalling pathways in hippocampus and prefrontal/frontal cortex of male rats. in the last several years, particular attention was given to camp-responsive element binding protein -related pathways, since it was shown that chronic antidepressants increase creb phosphorylation and transcriptional activity, through the activation of calcium/calmodulin-dependent and mitogen activated protein kinase cascades.
 the novel antidepressant agomelatine, a melatonergic mt1/mt agonist combined with 5-ht2c serotonin antagonist properties, showed antidepressant action in preclinical and clinical studies. there is a general agreement that the therapeutic action of antidepressants needs the activation of slow-onset adaptations in downstream signalling pathways finally regulating neuroplasticity. in the last several years, particular attention was given to camp-responsive element binding protein -related pathways, since it was shown that chronic antidepressants increase creb phosphorylation and transcriptional activity, through the activation of calcium/calmodulin-dependent and mitogen activated protein kinase cascades. aim of this work was to analyse possible effects of chronic agomelatine on time-dependent changes of different intracellular signalling pathways in hippocampus and prefrontal/frontal cortex of male rats. to this end, measurements were performed 1 h or 16 h after the last agomelatine or vehicle injection.  we have found that in naïve rats chronic agomelatine, contrary to traditional antidepressants, did not increase creb phosphorylation, but modulates the time-dependent regulation of mapk/erk1/ and akt/glycogen synthase kinase- pathways.  our results suggest that the intracellular molecular mechanisms modulated by chronic agomelatine may be partly different from those of traditional antidepressants and involve the time-dependent regulation of mapk/erk1/ and akt/gsk- signalling pathways. this could exert a role in the antidepressant efficacy of the drug. keywords antidepressantagomelatineintracellular signallingneuroplasticitytime-dependent regulationissue-copyright-statement© the author 2014 