| Schizophrenia is a serious mental illness,usually manifested as abnormal behaviors and mental models;Its complex pathogenesis prompts people to continuously explore its indepth pathological structure and effective therapeutic targets.The 5-HT1 a receptor(5-HT1 a R)in the serotonin(5-HT)system can be pointed out that it is closely related to schizophrenia.Although there were treatment drugs targeting 5-HT1 a R as early as the market in the 20 th century,its treatment effect was not significant and accompanied by part of the extrapyramidal symptoms.It is also because of the unique properties of 5-HT1 a R in different brain regions that there are heteroreceptorsand and autoreceptors,only systemic regulation of 5-HT1 a R cannot maximize its function.Extracelluar regulated properin kinase(ERK)is expressed in the central nervous system and participated in a variety of normal behaviors.It has been proved to participate in the pathological process of schizophrenia,but these studies and analysis are mostly concentrated on the whole brain.How to play an ERK pathway in a single brain area is not clear.The ventral subiculum(vSub)is the main structure of connecting the hippocampus and the enterhinal cortex(EC),and many other cortex.It has huge significance to the processing of information and emotional regulation.Excessive active in the vSub may be the key factor in schizophrenia.Therefore,according to the specificity of structure and function,it is particularly important to narrow the scope of regulation and find the area that can efficiently play.At present,there are few reports that 5-HT1 a R participates in the hyperactivity of vSub in schizophrenia-like model and regulates 5-HT1 a R in vSub to improve schizophrenia-like activity.So it is urgent to investigate the mechanism of 5-HT1 a R in vSub in schizophrenia-like activity.Objectives(1)Construction of MK-801-induced schizophrenia model in rats.(2)To assess the changes in 5-HT1 a R protein in the vSub of MK-801 rats.Modulating the 5-HT1 a R in the vSub on schizophrenia-like activity in rats and further exploring the changes in neuronal activity,5-HT1 a R protein and ERK pathway in this brain region.(3)Elucidating the effects of regulating the ERK pathway in the vSub on 5-HT1 a R protein expression.(4)Detection of projections between vSub and retrosplenial granular cortex(RSG).Methods(1)A schizophrenia-like animal model was constructed by intraperitoneal injection of MK-801 into SD(Sprague Dawley)rats,and the validity of the model was verified by behavioral experiments.(2)Rats were given intracerebral microinfusion of 5-HT1 a R antagonist WAY100635 or agonist 8-OH-DPAT bilaterally into vSub,and rats were given intraperitoneal injection of MK-801 or saline to form Saline+Saline,MK-801+Saline,MK-801+WAY100635 or 8-OH-DPAT,Saline+WAY100635 or 8-OH-DPAT in four groups.Behavioral experiments(including: open field test and prepulse inhibition)were performed on the rats for five consecutive days to assess the behavioral changes.The effect of modulating 5-HT1 a R in the vSub on neuronal activity in this brain region was detected using immunofluorescence techniques,and the excitatory neuronal marker Ca MKIIα was co-stained to analyze neuronal properties.Changes in the expression levels of 5-HT1 a R and p-ERK protein in the vSub after microinfusion treatment with 5-HT1 a R antagonists or agonists were detected using immunohistochemistry and western blotting(WB)technique.(3)Four groups of Saline+Saline,MK-801+Saline,MK-801+U0126,Saline+U0126 were formed by microinfusion of ERK pathway inhibitor U0126 into vSub of rats and intraperitoneal injection of MK-801 or saline,respectively.And the expression levels of 5-HT1 a R and p-ERK protein in the vSub were detected by immunohistochemistry and WB technique to elucidate the connection between ERK pathway and 5-HT1 a R.(4)Cholera toxin B subunit(CTB)was used to label the neural circuit between vSub and RSG by reverse tracing.And the properties of the projecting neurons were analyzed by detecting the degree of overlap between Ca MKIIα and CTB fluorescence using immunofluorescence technique.Results(1)A schizophrenia-like animal model was successfully constructed in rats induced by intraperitoneal injection of MK-801.Spontaneous activity was significantly enhanced in MK-801-induced rats.In the open field test(OFT),the distance and velocity of movement of MK-801 rats were significantly increased within 30 min(p<0.001).In the prepulse inhibition(PPI)test,the PPI% of MK-801 rats was significantly disrupted(p<0.001)and the amplitude of the startle response(ASR)was increased(p<0.001).(2)The 5-HT1 a R protein were significantly upregulated in the vSub of MK-801 rats.Microinfusion of the 5-HT1 a R antagonist WAY100635 into vSub significantly reduced MK-801-induced enhancement of schizophrenia-like spontaneous activity(p<0.05)and ameliorated MK-801-induced PPI% impairment(p<0.05).Further analysis revealed that antagonizing 5-HT1 a R in the vSub significantly increased the expression of c-Fos in this brain region(p<0.001)and overlapped with the excitatory neuronal marker Ca MKIIα(p<0.01),and decreased the expression of 5-HT1 a R and p-ERK protein(p<0.01).This indicates that WAY100635 most likely improved schizophrenia-like activity by modulating excitatory neurons and subsequently downregulating 5-HT1 a R and p-ERK protein expression,ultimately.The microinfused agonist 8-OH-DPAT,on the other hand,had no significant effect on MK-801-induced schizophrenia-like spontaneous activity(p>0.05),but exacerbated MK-801-induced PPI% impairment(p<0.05).Further findings revealed that agonism of 5-HT1 a R in the vSub also decreased MK-801-induced 5-HT1 a R(p < 0.001).8-OH-DPAT itself upregulated p-ERK levels,but had no significant effect on MK-801-induced increase in p-ERK.It indicates that 8-OH-DPAT can also regulate ERK pathway,but the regulation of ERK pathway is not significant under the injection of MK-801.(3)Antagonizing the ERK pathway in the vSub significantly reduced MK-801-induced the upregulation of p-ERK(p<0.001)and 5-HT1 a R(p<0.01)protein.It indicates that the ERK pathway also regulates 5-HT1 a R.(4)CTB reverse tracing shows the presence of excitatory neurons projecting to the RSG in the vSub.ConclusionsMK-801 induced schizophrenia-like activity in rats,such as enhanced spontaneous activity and impaired PPI%,while upregulating 5-HT1 a R and p-ERK protein levels in the vSub.By administering WAY100635,a 5-HT1 a R antagonist into vSub,the MK-801-induced enhanced spontaneous activity and impaired PPI% were reduced.This phenomenon may be caused by WAY100635 by acting on excitatory neurons and subsequently downregulating 5-HT1 a R and pERK protein levels.In contrast,microinfusion of the agonist 8-OH-DPAT exacerbated the phenomenon of impaired PPI% induced by MK-801.Agonism of 5-HT1 a R in the vSub also reduced the expression of 5-HT1 a R induced by MK-801(p<0.001),while 8-OH-DPAT itself upregulated the level of p-ERK,but had no significant effect on the increased p-ERK which induced by MK-801.It indicates that 8-OH-DPAT can also regulate ERK pathway,but the regulation of ERK pathway is not significant under the injection of MK-801.In addition,microinfusion of ERK pathway antagonist U0126 into vSub also reduced MK-801-induced 5-HT1 a R protein levels.This suggests that the ERK pathway and 5-HT1 a R can interact with each other.The above results suggest that in the vSub,5-HT1 a R antagonists but not agonists may improve MK-801-induced schizophrenia-like activity by acting on excitatory neurons and downregulating ERK phosphorylation pathway and 5-HT1 a R protein levels.At the circuit,excitatory neurons projecting to the RSG are present in the vSub,and the function of this circuit in schizophrenia-like activity remains to be verified. |
