Promotion On Neuronal Plasticity And The Underlying Mechanism Of The Antidepressant

Objective:Depression is a chronic, recurring and potentially life-threatening illness that affects up to20%of the population of the word. However the patho physio logy of depression is still not clear. Many studies revealed that BDNF and the neuronal plasticity were correlated with depression, however the specific and the correlation between them are still unknown. Single injection of ketamine could treat depression in30min and the effect could last for7days. However the underlying mechanisms are still unknown. To clarify the correlation among depression, BDNF and plasticity would let us know some more about the depression, and would support us new avenue to explore the novel antidepressants. Previous studies revealed that hederagenin showed effect on the treatment of depression, however, the biological activity is not very strong.To clarify the systematic correlations among BDNF, neuronal plasticity and depression. And then, one side, reconstruct the hederagenie to HGA, and test the biological activity on the treatment of depression, then studied the possible mechanism of HGA on the treatment of depression. Another side, make a study on the mechanism underlying the fast and long lasting antidepressant effect of ketamine, specially to reveal the correlation between the effect of ketamine and the variance of the BDNF and the synaptophysin. Methods:1. C57BL/6mice were exposured to chronic corticosterone to make the anxiety/depression-like model, elevated plus maze and tail suspension test were used to test the behavioral state of the mice after30days exposure of corticosterone. After the confirmation of the behavioral state of the mice. Mice were give fluoxetine(18mg·kg-1, ip) to reverse the anxiety/depression-like state. After20days treatment of fluoxetine, EPM and TST were used to test the behavioral state of the mice. During this period, mice were sacrificed at day10,20,35,45,60. The brains were fixed with4%paraformaldehyde solution and then the Golgi-cox-staining and immunofluorescence were carried out. For Golgi-cox-staining dendritic spines were scored from10-12neurons from each mouse in each subregion of hippocampus. For CA1and CA3, pyramidal neurons were selected for spine density evaluation, and for DG, granule neurons were selected for spine density evaluation. The spines were counted from the last branch point to the terminal tip of the dendrite. The dendritic spine density was counted at1000×(at least20μm in length) from different neurons in hippocampal subregions CA1, CA3and DG. For Immunofluorescence staining, images were acquired with a60×objective lens of a confocal microscope (FV10i, Olympus) in11subareas for each slice, and6-12cell bodies in each subarea were selected randomly for the measurement. The BDNF levels were expressed as a percentage of the vehicle group. An average value was obtained for each mouse for statistical analysis.2. The effect of ketamine on the expression of BDNF and the neuronal plasticity was studied. Chronic corticosterone exposure was recruited to make the depressive mice model, and then single injection of ketamine was carried out to study the different effect of ketamie on BDNF on depressive mice and normal mice by western blotting. On the basis of this, one side, whether the short-term exposure of corticosterone could affect the influence of ketamine on the expression of BDNF was studied, the other side, the pathophysiology underlying the fast effect and long-lasting effect of ketamine on the treatment of depression was studied. C57BL/6mice were exposured to corticosterone for3days then were sacrificed30min or7days after the single injection of ketamine (3mg·kg-1). The variation of BDNF and synaptophysin was detected by western blotting.Finally, the mechanism underlying the promotion of the neuronal plasticity of ketamine was studied. Racl-cofilin signalling pathway was studied after the single injection of ketamine. The variations of the related proteins. Total Racl, RaclGTPase, Tiaml, RacGAPl, cofilin and P-cofilin were studied by western blotting.3. The restructuring derivative of HG (HGA) was designed and synthesized. First the carboxyl group was activated by DCC/NHS, then the DMAPA (dimethylamino-1-propylamine) was added, and the aim product HGA was produced. Using mass-spectrography and NMR. After the thorough confirmation of HGA, HPLC was carried out to check the purity of HGA.Primary cultured hippocampal neurons from one day old SD rats were taken as the research object. The cell model was made by corticosterone in the primary cultured neurons. The protective effect of HGA on the injury induced by corticosterone was analyzed by MTT. The effect of HGA on the treatment of depression was analyzed by TST through acute drug delivery on C57BL/6mice.Results:1. The veriation of BDNF and neuronal plasticity during the procedures of the development and treatment of depression.(1) The effect of the chronic corticosterone exposure and long term treatment of fluoxetine on the behavior of the mice.After30days exposure of corticosterone, in the EPM, the number of entries into the open arms was significantly decreased after chronic corticosterone exposure in the corticosterone group compared with the vehicle group (t=2.202, P=0.043); in the TST, chronic corticosterone treatment induced a significant increase in the duration that the mice remained immobile compared to the vehicle group(t=2.094, P0.047). After25days treatment of fluoxetine (FLX), in the EPM, the number of entries into the open arms were significantly increased after20days treatment with fluoxetine compared with the corticosterone (CORT) group (P=0.007); while in the TST, the immobility duration decreased significantly compared with the corticosterone group (P=0.000).(2) The chronic effect of corticosterone and long term treatment of fluoxetine on the spine density of the neurons in the hippocampus.The Spine density was unchanged, following10and20days of corticosterone exposure in the hippocampal subregions CA1, CA3and DG, compared with corresponding vehicle group. After35days of corticosterone exposure, the spine density in CA1(t=2.626, P=0.030) and CA3(t=2.982, P=0.018) were significantly decrease compared with the vehicle group.45days of corticosterone exposure, significantly decreased the spine density in all subregions (CA1:P=0.004; CA3:P=0.000; DG:P=0.002) of the hippocampus compared with the vehicle group. Following60days corticosterone exposure, the spine density was also decreased in all of the neurons in the three subregions (CA1:P=0.001; CA3:P=0.000; DG:P=0.004), compared with the corresponding vehicle group. After10days treatment of fluoxetine, the spine density in CA1(P=0.000) and CA3(P=0.004) was significantly increased, compared with the corresponding CORT group. Following25days of fluoxetine treatment, spine density was significantly increased in all three subregions CA1(P=0.000), CA3(P=0.000) and DG (P=0.010), compared with the corresponding CORT group.(3) The effects of chronic corticosterone exposure and the long term treatment of fluoxetine on the quantity of BDNF in neuronal cell bodies in the hippocampusAfter10days exposure of the corticosterone, the expression of BDNF in mature neuronal cell bodies (t=3.371, P=0.010) and newborn neuronal cell bodies (t=4.416, P=0.002) in DG were significantly increased, compared with the corresponding VEH group. The expression of BDNF in neuronal cell bodies in CA3(t=3.495, P=0.008) mature neuronal cell bodies (t=3.848, P=0.005) and newborn neuronal cell bodies (t=3.991, P=0.004) in DG were significantly increased, compared with the corresponding VEH group. After35days of corticosterone exposure, the quantity of BDNF were increased significantly in mature neuronal cell bodies in the DG (t=2.501, P=0.037), CA3(t=3.670, P=0.006) and CA1(t=3.549, P=0.008) compared with the VEH group. After45days of corticosterone treatment, the quantity of BDNF were significantly decreased in the neuronal cell bodies in CA1(P=0.001) and the newborn neuronal cell bodies in DG (P=0.001) compared with the corresponding vehicle group. Following60days of corticosterone exposure, the quantity of BDNF in CA1(P=0.031),CA3(P=0.008) and the newbornal cell bodies in DG (P=0.012) were all increased.After10days fluoxetine treatment, the BDNF levels was significantly decreased in mature neuronal cell bodies (P=0.000) and new born neuronal cell bodies (P=0.003) in DG compared with corticosterone group. After25days of fluoxetine treatment, the levels of BDNF in the neuronal cell bodies of CA3, and the mature as well as the newborn neuronal cellbodies in DG were significantly decreased compared with the corticosterone group (CA3:P=0.025; DG-M:P=0.001; DG-N: P=0.001).2. The role of BDNF and neural plasticity in ketamine treatment in depression, and the mechanisms of strengthened neuronal plasticity induced by ketamine.(1) The role of ketamine on BDNF levels and the role of glucocorticoid corticosterone in the course of ketamine-induced BDNF expression.Compared with single injection of ketamine in normal mice, after30min, the administration of a single ketamine injection in depressed mice can make the expression of BDNF in hippocampus increased significantly (F=12.493, P=0.008). And the glucocorticoid receptor inhibitor Ru486can significantly inhibit this increased (F=15.499, P=0.004). But three days of corticosterone treatment can’t achieve a similar effect. Meanwhile, synaptophysin expression has no change after30min following a single ketamine in hippocampus (F=0.897, P=0.358). After7days, following a single injection of ketamine treatment, hippocampal BDNF expression was no longer affected in each group, it showed no significant change (F=1.539, P=0.233). Interestingly, synaptophysin expression was significantly increased after7days, following a single injection of ketamine treatment (F=466.104, P=0.000), and the Ru486pretreatment has no effect on the increasing expression of synaptophysin after7days following a single injection of ketamine treatment (F= 4.308, P=0.054).(2) The effect of ketamine on Racl signaling pathwayFollowing a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, the Racl-GTPase protein level increased significantly at15min (t=4.023, P=0.016) and45min (t=3.939, P=0.017) in hippocampus, and recovered to normal level at4h (t=0.570, P=0.599). Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, the Tiaml protein level increased significantly at15min (F=953.626, P=0.000) and45min (F=70.369, P=0.000) in hippocampus, and recovered to normal level at4h (F=1.259, P=0.278). Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, the RacGAPl protein level significantly reduced in ketamine treated mice at15min (F=20.750, P=0.000) and45min (F=181.808, P=0.000) time points. Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, inactive p-cofilin was significantly increased at15min (F=494.566, P=0.000),45min (F=207.595, P=0.000), and4h(F=119.280, P=0.000) time points, and return to basic levels at24h (F=2.453, P=0.137), following ketamine treatment.3. The study on the synthesis, structural characterization, the biological activity validation as well as the underlying mechanism of the HGA(1) The synthesis and the characterization of HGA By ordinary chemical synthesis and column chromatography separation, a white powdery compound was obtained. The Mass spectrum reveals the molecular ion peak is558.0([M+H]+), while the molecular weight of HGA is556.86. The1hNMR (CDC13) revealed:1H-NMR(CD3OD):δ5.26(t, J=3.4Hz,1H, C=C-H),3.99(dd, J1=14Hz, J2=7.2Hz,1H),3.48-3.52(m,1H),3.41(d, J=10.8,1H),3.05-3.11(m,1H),2.91(t,J=7.2Hz,2H),2.71(s,6H, NMe2),1.91(s,1H),1.05-1.43(m,22H),0.78-0.95(m,18H),0.686(3H, s, CH3),0.596(3H, s, CH3), and the datas are agreed with the structure of HGA. Data from13CNMR show that:δ180.02(C=O), 143.67(C=C),122.65(C=C),72.30,65.76,60.07,55.38,46.20,46.10,42.22,41.82,41.51,40.99,39.20,37.97,36.43,35.90,33.58,33.20,33.02,31.81,30.14,27.06,25.93,25.03,24.87,23.06,22.57,22.44,19.38,17.62,16.56,14.78,12.98,11.24. Based on the above analysis, the purity of HGA was checked by high performance liquid chromatography (HPLC) with methanol-water-acetic acid-the ethylenediamine (87:13:0.04:0.02) as the mobile phase,210nm as the detection wavelength. HPLC revealed that the peak time of HGA is about59.09min, and the normalization method results show that the HGA’s purity is94.08%(HPLC).(2) The antidepressant activity and the underlying mechanism of HGAIn the neuroprotective experiments of HGA, MTT results showed that the compound HGA (P=0.042) produced significant protective effect in a concentration of0.1μmol·L-1on the injury caused by corticosterone (10μmol·L-1) in primary cultured neurons, however, under the same experimental conditions, HG (P=0.866) did not have a significant protective effect on neurons at a concention of1μmol·L-1. The western blotting results show that the HGA can reverse the decline of expression of BDNF (P=0.000) and synaptophysin (P=0.000) caused by corticosterone exposure. And the results of the acute delivery of HGA (10mg·kg-1) in mouse tail suspension test revealed that HGA could significantly (P=0.010) decrease the immobility duration.Conclusions:1. During the occurrence and development of depression our data revealed an increase-decrease-increase fluctuation in the levels of BDNF. After treatment with antidepressants, fluoxetine reduced the stress-induced up-regulation of BDNF. Our data indicated that BDNF correlate with depression in a complicated increase-decrease-increase style.Dendritic spine density of hippocampal neurons decreased during the development of depression in mice, and increased after giving fluoxetine treatment. The impairment of dendritic spines in the hippocampus may underlie the development of depression. These results indicate that changes in spine density in the hippocampus maybe a pathophysiological mechanism underlying depression. 2. Ketamine could promote the BDNF expression significantly in depressed mice model compared with the normal mice, and such significant promoting effect can be blocked by the glucocorticoid receptor inhibitor Ru486. However, the short-term (3days) corticosterone treatment did not affect the ketamine’s promotion in BDNF expression. The expression of BDNF was significantly increased after30min following a single injection of ketamine, but the neuronal plasticity was not affected. After7days following a single injection of ketamine, BDNF expression returned to the basic level, while the expression of synaptophysin was significantly increased. The single dose ketamine improved neuronal plasticity may recruit Racl-GTPase signaling pathway, and ultimately increase the neuronal plasticity, to achieve the antidepressant effect.3. The hederagenin amide derivative HGA was designed and successfully synthesized. HGA can show protective effect on injuries induced by corticosterone in the primary cultured hippocampal neurons at a concention of0.1μmol·L-1. HGA could significantly decrease the immobility duration in tail suspension test by acute delivery. HGA can promote the expression of BDNF and synaptophysin which was reduced by corticosterone (10μmol·L-1).