Behavioral And Hippocampal Cytoskeletal Alterations In Rats Following Chronic Unpredictable Mild Stress And Fluoxetine Treatment

Background:Many findings strongly indicate an involvement of structural neuronal plasticity in the pathophysiology of stress and depression. The property of adult neurones to change their structure and function in response to new stimuli is called neuronal plasticity and includes changes in dendritic ramifications, synaptic remodelling, LTP, axonal sprouting, neurite extension, synaptogenesis and neurogenesis. Dynamic microtubules are fundamental for the remodelling and extension of axons and dendrites. Indeed, dynamic instability provides microtubules the capability of probing the intraneuronal space and rapidly paves the way to the eventual new synaptic partner. Microtubules are essential eukaryotic cytoskeletal organelles and growing evidence indicates that the microtubular system could play a role in stress-induced impairments in structural neuronal plasticity.Microtubules are formed by the polymerization of tubulin, a heterodimer of two subunits designated a andβ. Microtubules are functionally modified to several isoforms by post-translational modification.The expression of isoforms of a post-translational modification such as Tyr-Tub and Acet-Tub is currently used as markers of microtubule dynamics. Microtubules interact with microtubule-associated proteins (MAPs), which modulate polymerisation, stability and arrangement of microtubules. Indeed, when dephosphorylated, neuronal MAPs bind microtubules and promote tubulin assembly and microtubule stabilization. In contrast, phosphorylation of MAPs induces detachment and promotes microtubule dynamics.Antidepressant drugs have play an important role in knowing the etiology and mechanism of depression. It has been reported that chronic antidepressant treatment reverse the stress-induced decrease of hippocampal structural neuronal plasticity. Fluoxetine, as a typical antidepressant drug, is an index drug to valuate depression model. Findings have demonstrated that fluoxetine can reverse the stress-induced changes. Studies on the effects of antidepressant treatment upon the neuronal microtubular system are still in the early stages. However, current data indicate that both acute and chronic antidepressant treatments could exert an action on neuronal microtubular proteins and microtubule dynamics. Literatures indicate that a single administration of antidepressants can decrease microtubule dynamics, whereas the effect of chronic treatment such as fluoxetine enhance microtubule dynamics.There is growing evidence that depression may be best characterized as a chronic and recurrent disorder. Specifically, current estimates indicate that the rate of relapse/recurrent is very high. Approximately 50% of patients with major depressive disorder (MDD) experience recurrence, and relapse or recurrence rates are as high as 20-37% during the continuation or maintenance phase of pharmacotherapy. Prevention of recurrence and relapse is therefore one of the most important and challenging goals in the management of major depression. To the best of our knowledge, there have few literature published about the recurrence model of depression. Different from other studies, the rats in the present study were exposed to chronic unpredictable mild stress (CUMS) again after they recovered from the first CUMS induced depression, in order to simulate recurrence of depression.Objective:To investigate behavior and hippocampal morphology and cytoskeletal alterations following re-exposure to CUMS and acute swimming stress, and explore the possible mechanism.Method:(1) Animals were separated into one of five groups:control+vehicle (Control+V), chronic unpredictable mild stress+vehicle (CUMS+V), chronic unpredictable mild stress+fluoxetine (CUMS+FLX), chronic unpredictable mild stress+fluoxetine+drug washout (1 week)+acute swimming test(CUMS+FLX+AS), chronic unpredictable mild stress+fluoxetine+drug washout (1 week)+chronic unpredictable mild stress (CUMS+FLX+CUMS). (2) We used CUMS which has been shown to produce behavioral changes that are similar to human depression and considered to be a valid and useful experimental model of depression. (3) After received CUMS, rats of CUMS+V and CUMS+FLX were treated with vehicle. Rats of CUMS +FLX, CUMS+FLX+AS and CUMS+FLX+CUMS were treated with fluoxetine(10mg/kg). (4) Following fluoxetine treatment, rats of CUMS+FLX+ CUMS were re-exposed to CUMS to mimic the recurrence of depression to explore the possible alterations of cytoskeleton. Rats of CUMS+FLX+AS were re-exposure to acute swimming stress. (5) Increased weigh,24h ingestion, Sucrose preference test and open field test were assessed after CUMS, fluoxetine treatment and re-exposure to stress. (6) Nissl stain was used to observe the suvrival of the Pyramidal neurons in hippocampal CA1, CA3 and DG fields. (7) The expression of a-tubulin isoforms, MAP-2 and phospho-MAP-2 were analyzed used western blot.Results:(1) The 3 weeks of CUMS induced marked decrease in the Decreased weigh,24h ingestion, Sucrose preference test, traveled distance, moved velocity and frequencies of rearing in the stressed rats compared to the non-stressed group(p<0.01). At the end of fluoxetine treatment there were no differences between control and animals treated with fluoxetine for three weeks. In contrast, the behaviors of sucrose preference and open field were different between CUMS+V group and other groups (p<0.01 of all). There were no significantly change of sucrose preference, traveled distance, moved velocity and frequencies of rearing of CUMS+FLX+AS group following acute swimming stress. In contrast, animal treated with CUMS again consumed significantly less sucrose solution, traveled less distance and moved with less velocity in comparison with CUMS+V rats (p<0.01). The frequencies of rearing of CUMS+FLX+CUMS group were not statistically significant decreased compared to that of CUMS+V group (p=0.126). However, frequencies of rearing of CUMS+FLX+CUMS group were deduced to 0.125±0.354, lower than 3.75±2.053 of CUMS+V group. (2) The Pyrmaidal neurons in hippocampal CA1 region in CUMS+FLX+CUMS group were were singificantly decreased compare to other groups (p<0.01). The amount of neurons in hippocampal CA3 and DG region have no difference between CUMS+V, CUMS+FLX and CUMS+FLX+AS. The amount of neurons in hippocampal CA3 and DG region in CUMS+V was significantly less than that of Control+V group(p<0.01) whereas the amount of neurons in CUMS+FLX+CUMS was significantly less than that of CUMS+V group(p<0.01). (3) The densitometric analyses of the Acet-Tub expression of CUMS+V group showed a significant increase (P＜0.01) to 172±11% in rats submitted to CUMS and the Acet-Tub expression of CUMS+FLX+CUMS group increased significantly (P＜0.01) to 239±10% following re-exposure to CUMS. Furthermore, the post hoc analysis also showed there was significant deference between CUMS+V and CUMS+FLX+CUMS group (P<0.01, Bonferroni test). The densitometric analyses also showed that Tyr-Tub expression of CUMS+V group was significantly decreased to 61±11% following CUMS stress and the post hoc analysis revealed a significant (P<0.01) difference compared with other groups. Tyr-Tub expression of CUMS+FLX+CUMS group significantly decreased to 31 ±7% following re-exposure to CUMS, difference significantly (P＜0.01) compared with other groups, including the CUMS+V group. In contrast, the a-tubulin isoforms expression of animals treated with fluoxetine and animals received acute swimming stress did not differ statistically from the Control+V group (p>0.05 for both). The one-way ANOVA performed on levels of the hippocampal MAP-2 indicated that the staining of MAP-2 by MAP-2 antibody did not significantly alter after stress or re-exposure to stress while levels of the hippocampal phospho-MAP-2 altered significantly after stress and re-exposure to stress. The densitometric analyses of the phospho-MAP-2 expression showed a significant decrease (P＜0.01) to 64±9% in CUMS+V rats submitted to CUMS and a significant decrease (P<0.01) to 22±11% following re-exposure to CUMS in CUMS+FLX+CUMS group. In particular, the phospho-MAP-2 expression of CUMS+FLX rats and CUMS+FLX+AS rats did not differ statistically from the Control+V group (p>0.05 for both).Conclusion:Our results suggest that CUMS and fluoxetine affect microtubule dynamics in the hippocampus. These effects appear to be mediated by the degree of phosphorylation of MAP-2. Furthermore, the stressed rats were more sensitive to the subsequent CUMS and their hippocampal cytoskeleton became more impaired. It suggests a possible role of cytoskeletal proteins in mediating functional and/or morphological changes in rat hippocampus and in the recurrence/relapse of depression.