Hippocampal CA3 Calcineurin Activity Participates in Depressive-Like Behavior
in Rats
Wei-Li Zhu, Hai-Shui Shi, Shen-Jun Wang, Ping Wu, Zeng-Bo Ding, and Lin Lu*
National Institute on Drug Dependence, Peking University, Beijing 100191, China
*Corresponding author:
Prof. Lin Lu
National Institute on Drug Dependence
Peking University
38, Xue Yuan Road
Beijing 100191, China
E-mail: linlu@bjmu.edu.cn
Tel: +86-10-82802459
Fax: +86-10-62032624
Abstract: Objective Calcineurin is a serine/threonine protein phosphatase that
regulates neurotransmission, 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 CnA 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 g)  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
CnA 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 revealed 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 CnA 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 CnA 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.
Keywords: calcineurin; cyclosporine-A; mGluR2/3; depression; chronic mild stress;
hippocampus