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Objective Calcineurin is a serine/threonine protein phosphatase that regulates neurotransmissiou, neuronal structure and plasticity, and neuronal excitability in mood disorders, including depression.Increasing evidence has suggested that calcineurin is involved in the regulation of depressive-like behavior.However, little is known about the neurobiological mechanisms that underlie the mood-regulating effects of calcineurin.In the current study, we aimed to investigate the potential mechanism by which calcineurin mediates the development of depressive-like behavior and the involvement of calcineurin in the action of antidepressant medication in the chronic mild stress (CMS) model.Methods Rats were subjected to stress daily for 3 weeks and the hippocampal tissues were removed for the measurement of calcineurin activity by assessing CnAα and pSynapsin Ⅰ ser62/67 levels using Western blot.Pharmacological cyclosporin-A (CsA) used to inhibit calcineurin activity selectively in CA3 of rat hippocampus to determine the role of calcineurin in behaviors related to depression.To assess the effect of calcineurin inhibition on the antidepressant effect of venlafaxine, rats were infused with DMSO or CsA into the CA3, and venlafaxine (40 mg/kg) was administered intraperitoneally (i.p.) 30 min after CsA infusion once daily, subsequently, the sucrose preference test was performed to measure antidepressant efficacy.We then determined whether the increase in depressive-like behavior induced by calcineurin inhibition could be normalized by mGluR2/3 activation.LY354740, a highly selective group Ⅱ mGluR agonist, was used to measure the effect of activated mGluR2/3 on depressive-like behavior induced by the calcineurin inhibitor.Four groups of rats (n =9-10 per group) in a 2 (CsA dose: 0 and 2 μg) × 2 (LY354740:0 and 100 ng) factorial design were used in the forced swim test.CsA or its vehicle was microinjected into the CA3 30 min before the forced swim test.LY354740 or its vehicle was microinjected into the CA3 10 min before CsA administration.Finally, to determine the possible role of calcineurin on antidepressant action.Three groups of rats (n =8-11 per group) were treated with vehicle, venlafaxine (40 mg/kg), or haloperidol (1 mg/kg) intraperitoneally for 21 consecutive days.Thirty minutes after the last drug administration, the animals were immediately decapitated, and brains were removed for subsequent pSynapsin and CnAα level analysis using the Western blot assay.Results The results revealed that CMS increased pSynapsin S62/S67 in the CA3 without alterations in total synapsin Ⅰ levels, suggesting that calcineurin activity was decreased in CA3.However, no significant changes in pSynapsin and Synapsin levels were found in the CA1 or DG after CMS.These data suggest that CMS inhibited calcineurin specifically in the CA3 region of the hippocampus.We also found that depressive-like behavior induced by CMS is associated with decreased calcineurin activity in the CA3 but not in CA1 or DG of the hippocampus.Microinjection of CsA into the CA3 decreased mGluR2/3 protein level, induced depressive-like behavior in normal rats, and exacerbated depressive-like behavior in CMS-treated rats.DG calcineurin inhibition neither induced depressive-like behavior in control rats nor exacerbated depressive-like behavior in CMS rats.Additionally, the behavioral effects of the antidepressant venlafaxine were reversed by administration of the calcineurin inhibitor CsA in the CA3.The findings suggest the specific role of CA3 calcineurin activity in the deleterious effects of chronic stress and the therapeutic effects of antidepressant treatment.Moreover, infusions of the mGluR2/3 agonist LY354740 into the CA3 reversed the depressive-like behavior induced by CsA administration in the forced swim and novelty-suppressed feeding tests.In the forced swim test, microinjection of the calcineurin inhibitor CsA into the CA3 increased immobility compared with the vehicle group.Importantly, this depressive-like behavior was reversed by pretreatment with the mGluR2/3 agonist LY354740.We also found that neither CsA nor LY354740 altered locomotor activity, reflected by crossings and rearings assessed in the open-field test.These data suggest that calcineurin inhibition induced a depressive-like phenotype that could be reversed by treatment with a mGluR2/3 agonist, with no effects on locomotor activity in rats.In the novelty-suppressed feeding test, intra-CA3 infusion of CsA significantly increased the latency to feed.We then determined whether activation of mGluR2/3 was able to reverse this increase in the latency to feed induced by calcineurin inhibition.The results reveaIed that the increase in the latency to feed in CsA-treated rats was reversed by pretreatment with the mGluR2/3 agonist LY354740, without altering home cage food consumption..Altogether, these results suggest that activation of mGluR2/3 is effective in reversing depressive-like behavior induced by calcineurin inhibition.Finally, chronic venlafaxine treatment decreased pSynapsin S62/S67 levels in the CA3, whereas venlafaxine did not alter pSynapsin in the CA1 or DG.Furthermore, chronic venlafaxine treatment increased CnAα levels in the CA3 but not CA1 or DG.However, chronic haloperidol treatment had no effect on pSynapsin levels in the CA1, CA3, or DG and did not change CnAα levels in these three regions.These results indicate that the antidepressant had a specific effect on calcineurin activation selectively in the CA3.Conclusion These findings suggest that CMS decreased CA3 calcineurin activity.Calcineurin inhibition in the CA3 but not DG induced depressive-like behavior and reversed the antidepressant-like effect of venlafaxine.Activation of CA3 mGluR2/3 normalized the depressive-like behavior induced by the calcineurin inhibitor.Our findings might contribute to the development of novel antidepressant interventions that target specific calcineurin and downstream glutamatergic neurotransmitter systems to achieve therapeutic benefits.