| Schizophrenia (SCZ) is a serious psychotic disease which affects about 1% of the world population. SCZ patients suffer from positive symptom (delusions, auditory hallucinationa and thought disorder) and negative symptom (blunted affect and emotion, alogia, anhedonia and avolition) accompanying with deficits of cognitive and these symptoms become apparently at late adolescent time or in early adulthood. Patients with negative symptoms appear to have a poor quality of life and lead to the burden on relative.Schizophrenia is reported to be related to abnormal development of cortex which was caused by genes and environmental factors. Most SCZ patients have mental and behavioral abnormalities at an early age; adverse factors, during the perinatal and neonatal period, could lead to SCZ in the adult period; minor physical anomalies are related to SCZ. These evidences support that SCZ is related to development of neural system. In the brain, frontal cortex is the largest brain region and regulates biological functions including cognitive, emotion, social interactions and so on. Generally, frontal cortex is believed to be responsible for negative symptom in SCZ patients. Neuropathological studies confirm that SCZ patients had thinner cortex and reduced nerve fibers in early childhood; animal researches reveal that malformation of cortical development could results in SCZ. Clinical researches address that schizophrenia were correlated with incorrect parental monitor. Based on researches, scientists formulat the neurodevelopmental hypothesis:adult SCZ was a consequence of disturbed development of cortex which caused by genetic or environmental factors. And it is important to elucidate the influence of disturbed development of cortex on the SCZ.Researches reveal that the pathology of schizophrenia is related to neurotransmitters including dopamine, serotonin, and glutamate system. In the past decades, most scientists pay attention to dopamine system in SCZ, however, negative symptom could not be clearly explained by disfuction of dopamine system. Now plenty of evidence supported that disfunction of N-methyl-D-aspartic acid receptor (NMDA) receptors . takes part in pathology of schizophrenia. Non-competitive NMDA receptor antagonists such as dizocilpine (MK-801) are well known to result in psychotic states including positive and negative symptoms in human. Systemic administration of MK-801 produces schizophrenia-like behavioral, cellular and molecular manifestations which are similar with manifestations in the patients with schizophrenia. Disfunction of frontal cortex is found in the schizophrenia patients. Reduced dendritic spine density and number of dendrite, on prefrontal cortical pyramidal neurons, are found in both SCZ models and SCZ patients. In the animal model of schizophrenia, administration of MK-801 could lastingly enhance frontal cortex activity and damage the neural network of prefrontal cortex which played a role in social interactions. MK-801 could also induce neuronal apoptosis both in vivo and in vitro. MK-801 could cause apoptosis of neurons in several brain regions in the primate. These evidences support that disfunction of NMDA receptor are in SCZ models and patients.NMDA receptors performed a critical role in the development of rodent brains. During the first 2 weeks, establishment of synaptic connections, increase of brain volume and proliferation of neurons occurs in rodent animals. At the same time, NMDARs are highly expressed in the neonatal mice and the rodent brain is vulnerable to NMDA receptors antagonism. Chronic administration of MK-801 to neonatal mice could induce deficits in behaviors including spatial learning deficits and working memory deficits which manifests in schizophrenia patients. This kind of animal models combines the disfunction of NMDA receptors with disturbed development of cortex, and is widely used in the researches. However, the underlying molecular mechanism of the model is unclear; social interaction alterations in adulthood are not clearly elucidated; effects of atypical antipsychotic drugs on the model are not revealed.MicroRNAs, a small non-coding RNA, could induce RNA silencing and post-transcriptional regulation of gene expression. Many microRNAs contribute to neural development and psychiatry. MiR-134 is a brain-specific microRNA and also regulates neuroplasticity and neural development. Both NMDAR and miR-134 are highly expressed in the dendritic spines during the first two weeks, and NMDA could increase the expression of miR-134. Decreased expression of miR-134 is found in the frontal cortex and hippocampus in a mouse model of schizophrenia. Convincing evidence comes from postmortem analysis of schizophrenia patients, miR-134 expresses abnormally in prefrontal cortex Brodmann area 46, which suggests miR-134 participates in the pathogenic mechanism of schizophrenia.SIRTuinl (SIRT1), acting as a NAD-dependent class III deacetylase in the nucleus, is an important regulating gene influencing many genes. SIRT1 is highly expressed in embryo mediating the branch of dendrites and neurite development. During the embryonic period, SIRT1 could promote the neurite development in hippocampus. NMDAR antagonism could attenuate the down-regulated expression of SIRT1 induced by glutamate. Especially, SIRT1 is reported to be related to schizophrenia of Japanese population. Although both SIRT1 and miR-134 are related to schizophrenia, limited study is available on the effects of them on schizophrenia induced by neonatal administration of MK-801.Paliperidone, approved by FDA, is the second generation antipsychotic drugs and the active metabolite of atypical antipsychotic risperidone. Paliperidone could restore the symptoms of schizophrenia, especially negative symptom. Clinical studies demonstrate that paliperidone attenuated the social interaction deficits. Current researches suggest that paliperidone could offer neuroprotective effects. For example, paliperidone protected SH-SY5Y cells against cell death induced by β-amyloid peptide (25-35). In addition, paliperidone reduced the caspase-3 expression and protected SK-N-SH cells against neurotoxicity induced by dopamine. Our previous study also suggested that paliperidone could protect primary cortical neurons against apoptosis induced by MK-801. Current therapeutic researches address that paliperidone not only have neuroprotective effects, but also ameliorate the negative symptom. Effects of paliperidone on the damaged neruons and social interaction deficits induced by MK-801 were not clearly addressed.Basd on above questions, we have designed our studies as follows:in the part I, we investigated effects of paliperidone on the MK-801-injured neurons and underlying mechanism; in the part Ⅱ,we explore the effects of paliperidone and mechanism underlying reduced excitabiliy and synaptic communication of models through patch clamp and electroporation; in the part Ⅲ, effects of paliperidone on social interaction deficit induced by postnatal administration of MK-801 were revealed.Part Ⅰ Neuroprotective effects of paliperidone on the MK-801-injured primary cultured cortical neuronsFirstly, we performed the assessment of MK-801 induced toxicity and dose response. Primary cultured neurons grew with more neurites; but treated with MK-801, morphological alterations of cells were observed under phase contrast microscope. Three different concentrations of MK-801 (50,100 and 200 μM) were added into the medium for 24 h, and then a dose-dependent toxicity on cells were evident. In summary, the disappearance of neurites increased, and the shape also changed to ellipse.50 μM Mk-801 resulted in a slightly damages of cells, while 100 and 200 μM MK-801 resulted in severe cell damages. We examined the influence of MK-801 on cell viability using CCK-8 assay. After treated with 50 μM MK-801 for 48 h, cell viability decreased to 85.72±4.17% of the control, however, in the 100 and 200 μM MK-801 groups, cell viability decreased significantly to 80.35±2.41% and 60.24±3.77% of the control. In our experiment, statistical significant differences were obtained only at 100 and 200 μM MK-801 (p< 0.01 and p< 0.001). To further detect the cell viability, we explored LDH release rates between different groups. The LDH release rates of neurons exposed to MK-801 (Control,50,100 and 200 μM) were 0.30±0.02, 0.40±0.01,0.45±0.02 and 0.56±0.02. Exposure to 100 and 200 μM MK-801 significantly changed the LDH release rates (both p< 0.01) but not 25 and 50 μM. Treatment with 100 and 200 μM MK-801 could affect the viability of primary cultured neurons measured by both CCK-8 assay and LDH assay. And 100 μM MK-801 was used in the following experiments.Furthermore, to examine whether the paliperidone has neuroprotective effects, we evaluated MK-801 injured primary cultured neurons companied with or without the paliperidone using the CCK-8 and LDH assay. In the presence of MK-801, three different concentrations of paliperidone (1 μM,10 μM, and 50 μM) were added into the medium. In the group with 1 μM and 50 μM, primary cultured neurons had less neurites. However, the number of neurites increased obviously and most cells were alive in the group with 10 μM paliperdione comparing with 50 and 1 μM.The CCK-8 assay and LDH release assay were used to explore the neuroprotective effect of paliperidone in the experiment. In the CCK-8 and LDH tests, only 10 μM paliperidone demonstrated neuroprotective effect in the presence of 100 μM MK-801 (p< 0.05 and p< 0.01). These results showed that a dose dependently protection of paliperidone against MK-801 induced primary cultured neurons death.10 uM paliperidone was used in the following examinations.In order to detect paliperidone’s effects on electrophysiological characteristic of primary cultured neurons, patch clamp was used. We detected the excitability of neurons in the MK-801 treated group and paliperidone treated group. After treatment of MK-801 for 48 h, there was no action potential, which was elicited somatic current injection. Paliperidone could reverse this effect. And then we compared peak amplitude and frquency of action potentials. Paliperidone counteracted the reduction of peak amplitude and frequency of evoked action potentials induced by MK-801.We explored the effects of MK-801 on Ih currents in primary cultured cortical neurons which were treated with different drugs. Voltage clamp technique was performed, and Ih currents were evoked by a holding potential from-60 to-140 mV. Application of MK-801 increased the amplitude of Ih currents to a level higher than control group (p< 0.01). Paliperidone did reverse the influence induced by MK-801 (p < 0.05).Finally, to examine the potential mechanism of paliperidone on primary cultured cortical neurons against MK-801 neurotoxicity, expression of SIRT1 and miR-134 were accessed by quantitative real-time PCR and Western blotting. The application of MK-801 led to a significant decrease in mRNA expression level of SIRT1 (p< 0.001). We also observed paliperidone significantly increased the expression level of SIRT1 (p < 0.05). The protein expression of SIRT1 was analyzed by Western blotting. Compared with the MK-801 injured group, paliperidone resulted in a significant increase in SIRT1 level in presence of MK-801 (p< 0.01).Because the expression of miR-134 was inhibited by SIRT1, we used quantitative real-time PCR to detect the expression level of miR-134. Treating with MK-801 lead to increased expression of miR-134, and paliperidone significantly reversed this trend (p < 0.05). These results showed that paliperidone could increase SIRT1 level and down-regulate the expression of miR-134 in the presence of MK-801.To explore if the neuroprotective influences of paliperidone was regulated by SIRT1, primary cultured cortical neurons were treated with the SIRT1 inhibitor nicotinamide for 24 h in presence of MK-801 and paliperidone. Application of nicotinamide resulted in a decrease in the number of neurites and cells compared with paliperidone-treated groups. Furthermore, CCK-8 assay also revealed that nicotinamide decreased the viability of primary cultured cortical neurons in the presence of MK-801 and paliperidone (p< 0.05). The result of LDH was similar to that of CCK-8 assay (p< 0.05). All these results suggested that nicotinamide could repress the protective effects of paliperidone through inhibiting the function of SIRT1. mRNA expression of miR-134 were inhibited by transfection, and excitability of neurons was accessed by patch clamp. The results revealed that down-regulated expression of miR-134 increased the cell viability.Part Ⅱ Paliperidone protects frontal cortex neurons against MK-801 induced inhibition of excitability in postnatal mouseIn order to investigate the influences of injection of paliperidone and MK-801 on the neurons, patch clamp was used to record the electrophysiological properties of neurons in the frontal cortex. We compared the spontaneous firing and intrinsic membrane properties of neurons in the frontal cortex after injection of MK-801 without/with paliperidone. Lower frequency and smaller amplitude of spontaneous firing were found in the MK-801-treated group, but paliperidone restore the frequency and amplitude (p< 0.05 and p< 0.05). Membrane resistances of neurons showed no significance among three groups; however, the membrane time constant of MK-801 group was bigger than the control (p< 0.05). Paliperidone attenuate the tendency (p> 0.05 and p< 0.05). We also observed bigger amplitude and higher frequency of spontaneous actin potentials of paliperidone-treated group than those in MK-801-treated group (both p< 0.01). Resting membrane potentials of neurons were not different (p> 0.05).In order to further assess influences of MK-801 and paliperidone on neurons in frontal cortex, current injections were made to evoke action potentials. Neurons displayed different evoked action potentials after injections of MK-801 with/without paliperidone. MK-801 decreased the amplitude and increased half-width duration of evoked action potentials (both p< 0.01), injection of paliperidone caused a significant increase in the amplitude and a decrease in half-width duration of evoked action potentials (p< 0.05 and p< 0.01).In order to further investigate effects of MK-801 on synaptic activity, we recorded sIPSC of neurons in frontal cortex. Neurons were held at-70 mV, sIPSCs were recorded at the presence of CNQX and D-AP-5. We compared the sIPSCs recorded from different groups. MK-801 lowered the amplitude and frequency of sIPSCs (p< 0.05 and p< 0.001). Compared with MK-801-treated groups, paliperidone increased the amplitude of sIPSC.To explore the possible molecular mechanism of paliperidone on postnatal day 7 mice treated with MK-801 (0.5 mg/kg), SIRT1 and AKT expression were accessed by western blot. Administration of MK-801 significantly lowered the expression of SIRT1, and paliperidone reversed the effect. Phosphorylation of AKT was inhibited in MK-801 treated group, and paliperidone raised the phosphorylated expression level of AKT (p< 0.05). Expression level of miR-134 was quantitatively assessed by real-time RT-PCR. Paliperidone could decrease the miR-134 expression compared with MK-801-treated group (p< 0.05).Finally, prefrontal cortical neurons were transfected with shRNA vector by using electroporation. In shRNA-SIRT1 group, SIRT1 expression was repressed by shRNA; in shRNA-miR-134 group, miR-134 expression was inhibited by shRNA. No spontaneous firing was detected in both shRNA-SIRT1 and control group. Amplitude of evoked action potentials was reduced in shRNA-SIRT1 group compared with control group (p< 0.01). Amplitude and frequency of sIPSC were lowered in the shRNA-SIRT1 group (p< 0.05 and p< 0.01). And amplitude and frequency of mEPSC were lowered in the shRNA-miR-134 group. (p< 0.05 and p< 0.01), compared with control group. Amplitude and frequency of action potentials were not influenced by shRNA-miR-134.Part Ⅲ Paliperidone attenuates the social interaction deficits induced by postnatal administration of MK-801Social interaction, in this experiment, was divided into a contact event, a relative position event, escape events and stop events. The contact event was defined as any distance which between 2 mice is< 1 cm; the relative event was defined as any distance that between 2 mice is< 3 cm; escape event was defined as a procedure in which 1st mouse goes to 2nd mouse and 2nd mouse escapes threshold in and out.We used adult mice model of SCZ, which had been widely used in the experiments, to employ whether MiceProfiler could apply to assessment of social deficits. MK-801 significantly reduced the number and duration of contact event, relative position events and stop-state in MK-801 treated mice, and paliperidone could partly counteract these effects. These results were coincided with previous studies and proved that MiceProfiler could be used to employ the social deficits of SCZ.Social deficits were analyzed by MiceProfiler in the mouse models induced by postnatal injection of MK-801. Contact events and relative position events are important component of social interaction. The social behavior repertoire of different drugs injected mice was captured by a computerized video and then analyzed by MiceProfiler software. MK-801 obviously reduce the number of contact events during 0-4min and 4-8min (p< 0.001 and p<0.01), but paliperidone significantly restored the number of contact events. During 0-4min and 4-8min, the number of relative position event was remarkably decreased in MK-801 treated mice (p< 0.05 and p< 0.001), paliperidone partly counteract these effects. It showed that MK-801 injected mice were not willing to keep in near position with others, but paliperidone could enhance the willing.We also used MiceProfiler to explore the effects of MK-801 on the number and duration of escape events. MK-801 increased the number and duration of escape events during 0-4min (both p< 0.001), then we observed significant reductions in the number and duration of escape events in the paliperidone-treated group (both p< 0.05). Moreover, MK-801 treatment produced similar results during 4-8min. MK-801 could decrease the number of stop-states during 0-4 min and 4-8 min (p< 0.01 and p< 0.05), and paliperidone tended to attenuate the effects. The information from the above repertoire indicated that MK-801 treated mice were afraid to contact with other mouse, either they had lower motivation in social interaction; however, paliperidone could attenuate this deficits.ConclusionPathogenesis and treatment of schizophrenia were hot topics in related research area. But effects of postnatal MK-801 administration on excitability of neurons and social interaction deficits were not revealed. In our studies, primary cortical neurons were treated with MK-801 and/or paliperidone and we found paliperidone protected neurons against MK-801 induced neuronal damage through regulation of SIRT1/miR-134. Secondly, we found MK-801 could inhibit neuronal excitability and synaptic functions through reducing amplitude of spontaneous and evoked action potentials and sIPSCs, but paliperidone could retard inhibtion induced by MK-801. Through silent gene expression using iontoporation, we also reveal that SIRT1/miR-134 take part in the inhibition of excitability and synaptic function induced by MK-801. Finally, a video-based software, MiceProfiler, was used to analyze social interactions in adulthood induced by postnatal MK-801 administration, and we revealed that paliperidone could attenuate social interaction deficits. In conclusion, the study addressed that paliperidone attenuated MK-801-induced neurotoxicity, inhibition of excitability and social interaction deficits in adult mice. |
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