Regulation Of Antidepressants On The Hippocampal Cytoskeleton

In persons suffered from several stress-related neuropsychiatric illnesses and in experimental animals exposed to stress, some structural alterations could be found such as atrophy of the apical dendrites of CA3 pyramidal cells and suppression of adult neurogenesis in the dentate gyrus (DG). Several kinds of antidepressant could reverse the structural and functional alterations induced by depression by modulating different signal transduction pathways. The cytoskeleton is a complex meshwork consisting of microtubules, actin microfilaments, intermediate filaments, and other associated proteins. Modifications in the neuronal cytoskeleton underlie the morphological changes including process outgrowth, neurite arborization, synapse formation and so on. Neuronal morphology is known to be highly responsive to extracellular and intracellular signals. Although these studies suggested a possible involvement of cytoskeleton in the pathogenesis of depression and antidepressant's action, the actual mechanisms were still unknown. Thus, the aim of this study is to examine whether cytoskeleton and related signal transduction are involved in the structural changes in stressed animals described in the hippocampus of depressed animals and how antidepressants regulate them.1. Effect of destroyed stabilization of microtubule on the behavior and antidepressant effect of rats.Microtubule is a prominent component of the cytoskeleton that plays an important role in neuronal maturation and maintenance the shape of nervous cells. Colchicine is a potent and specific microtubule-depolymerizing agent which binds tightly to theβ-tubulin subunit of theα/β-tubulin heterodimer, thereby decreasing the soluble tubulin pool and inhibiting microtubule assembly. Taxol is a microtubule stabilizing agent derived from the plant Taxus brevifolia. It is known that, by binding to tubulin at a ratio of 1:1, taxol stabilizes the microtubules by inhibiting the dissociation of tubulin dimer at the ends. In the present study, intradentate injection of colchicine/or taxol was used to destroy the stability of microtubule of hippocampal cells, and the role of microtubules in the antidepressant's action were examined.Firstly, rats were randomly divided into several groups: vehicle1 +NS/drugs (hippocampal infusion of vehicle + i.g.or i.p. NS/drugs), COL+ NS/drugs ( hippocampal infusion of colchicine + i.g.or i.p. NS/drugs ), vehicle2 + NS/drugs (hippocampal infusion of vehicle + i.g.or i.p. NS/drugs), taxol + NS/drugs ( hippocampal infusion of taxol + i.g.or i.p. NS/drugs). In each hippocampus, colchicine (0.7μg/μL, 2μL/site) or taxol (0.7μg/μL, 2μL/ site) was injected into the DG of adult wistar male rats.1) Effect of desipramine (DMI) on the immobility time after hippocampal microfusion in forced swimming test. 7 days after lesion surgery, open-field locomotion showed that intradentate injection of colchicines or taxol had no significant effect on open-field locomotion compared with vehicle group, respectively. Then with the model of forced swimming test, the effect of DMI(30mg/kg, i.p.) on the immobility time after hippocampal microfusion for 9 days was measured. DMI significantly decreased the immobility time in comparison with the vehicle +NS (P < 0.05). But after hippocampal infusion of colchicine, the antidepressant-like effect of DMI was disappeared; and the similar results were obtained in the animals with hippocampal infusion of taxol.2) Effect of concomitant treatment with fluoxetine(FLU) on the behaviour after hippocampal microfusion. 10 days after hippocampal infusion of colchicine/taxol, rats were administrated with FLU (10 mg/kg, i.g.) for 15 days. Then, novelty-suppressed feeding test and forced swimming test were used to evaluate the effect of FLU on the rats via hippocampal infusion. After 15-day treatment with FLU, the latency to feed of rats via hippocampal infusion of vehicle (vehicle + FLU) was decreased compared with vehicle + NS group, while the latency of COL+ FLU group and taxol + FLU did not differ from that of the COL + NS group or taxol + NS group, respectively. Interestingly, the latency to feed was increased to rats via hippocampal infusion of colchicine (COL + NS) compared with vehicle group (vehicle +NS), and the similar results were obtained in hippocampal infusion of taxol. After 15-day treatment with FLU, the latency to feed of rats via hippocampal infusion of colchicine (COL + FLU) was also increased compared with vehicle + NS group, but did not differ from that of the COL + NS group. No difference was found between taxol + FLU group and taxol+ NS group. After treatment with FLU for 15 days, with the model of forced swimming test, the immobility time of vehicle + FLU group was decreased than that of the vehicle + NS group, while the immobility time of COL + FLU and taxol + FLU did not differ from that of the COL + NS group or taxol + NS group, respectively.2. Effect of chronic unpredictable mild stress and concomitant treatment with antidepressant on the level of microtubule associated proteins in the rat hippocampusMicrotubules contain the core protein tubulin and variety of microtubule-associated proteins (MAPs), which co-assemble with microtubules and stabilize them. The best-studied MAPs are the MAP-2 and Tau proteins. MAP-2 is an abundant component of the neuronal cytoskeleton whose function is related to the outgrowth and stability of neuronal processes, and synaptic plasticity. MAP-2 consists of five subunits, MAP-2A, MAP-2B (both having an apparent Mr 270～280kDa) and MAP-2C, MAP-2D, MAP-2E (Mr 70～75kDa). MAP-2 is extensively phosphorylated, and the phosphorylation state of MAP-2 modulates its function and metabolism. Despite extensive evidence of a correlation between the expression of (p)-MAP-2 and neuronal development and plasticity, little is known of the relationship between (p)-MAP-2 and depression. In this study, the monoclonal antibody AP-18 was used which could bind MAP-2A/B and C, but only when Ser136 is phosphorylated.Firstly, rats were exposed to the chronically mild stress (CMS), an experimental model of depression, which has been used for screening the antidepressant activity of drugs. CMS caused a gradual decrease in the consumption of 1% sucrose solution and resulted in a decrease of open-field behaviour, and concomitant treatment with FLU could reverse these changes.Then, the change of (p)-MAP-2 was evaluated by immunohistochemistry. Compared with the control, rats received chronic stress showed significantly decreased immunostaining for the (p)-MAP-2. Concomitant treatment with FLU (10 mg/kg, i.g.) could increase the level of (p)-MAP-2.Immunohistochemical staining for (p) MAP-2 suggested that there was a decrease in overall intact protein. So, we then used western blot to explore the changes of subunits which showed that chronic stress significantly diminished (p)-MAP-2, especially the level of the low molecular weight (p)-MAP-2C. In addition, there was no marked change in the level of Tau protein in the DG after chronic stress or concomitant treatment with FLU.In particular, MAP-2 is predominantly expressed in neurons and serves as substrates for most of protein kinases, such as extracellular signal-regulated kinase (ERK). So, we next further detected the change of (p)-MAP-2 related signaling pathway kinase. We also found that the chronic stress down-regulated the level of pERK/ERK protein expression in hippocampus, while concomitant treatment with FLU (10 mg/kg, i.g.) reversed these changes. When exposed to PD98059 (a specific inhibitor of the mitogen-activated protein kinase kinase) 10～20μmol/L, the level of pERK was decreased, meanwhile, the level of p-MAP-2C was also decreased signicantly in a dose-dependent manner. The level of p-MAP-2A of PD98059-treated groups did not differ from that of the control group. With the method of immunocytochemistry, we can also find the length of prosess in cultured neuron was decreased significantly in the present of PD98059.3. Effect of chronic unpredictable mild stress and concomitant treatment with antidepressant on the level of glial fibrillary acidic protein (GFAP) in the rat hippocampusUsing a chronic psychosocial stress paradigm, we found a significant decrease of GFAP in response to stress. And concomitant treatment with FLU can block this effect. As we know, GFAP is the marker of astrocytes which synthesize and release many neurotrophic factors vital for neuronal health such as vascular endothelial growth factor (VEGF). The decrease of this protein may lead to impairment, reducing the levels of VEGF which regulates neuronal growth, maintenance, and plasticity. Chronic stress could reduce the expression of VEGF in the hippocampus, which in turn can be prevented by long-term chronic FLU treatment. In vitro, VEGF(10～20ng/mL) could defend cultured hippocampal neurons from neurotoxicity induced by corticosterone(100μmol/L). The similar result was obtained in cultured hippocampal progenitor cells. Treatment with VEGF(10～20ng/mL) significantly increased the proliferation of cultured hippocampal progenitor cells dose-dependently.Conclusion:1) Intradentate injection of colchicines or taxol induced depression-like behavior of rats and blocked the behavioral effects of acute/or chronic antidepressant treatment.2) Hippocampus of adult rat brain could also express MAP-2C; Chronic unpredictable mild stress could decrease the level of (p)-MAP-2 and concomitant treatment with antidepressant could reverse these changes; P-ERK was one of the most important kinases to regulate the phosphorylation of MAP-2C, and also, MAP-2C played a vital role in neurite outgrowth and cell shape regulation.3) VEGF played important roles in neuroprotection and neruogenesis, and it was likely that the down-regulation of the cytoskeletal GFAP protein induced by chronic unpredictable mild stress contributed to the impairment of VEGF.