| Background:Parkinson’s disease(PD)is one of the most common neurodegenerative diseases,with the main pathological characteristics of selective and progressive loss of dopaminergic(DA)neurons in the dense part of the substantia nigra,formation of Louis bodies(LB)in neurons and glial cell proliferation.The mouse injury model induced by neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)is one of the most commonly used PD animal models.However,there is still controversy over whether the MPTP-induced PD mouse model exhibits significant motor and emotion-related non-motor disorders.Some PD patients often have anxiety symptoms before the onset of exercise symptoms,which has seriously affected the quality of life of patients.However,the existing treatments have no significant effect on the emotional symptoms,and the mechanism and neural circuits involved in PD with anxiety are still unclear.The ventral tegmental area(VTA),the center of the limbic circuit of the midbrain cortex,is involved in the expression and regulation of anxiety by forming functional connections with multiple brain regions,which connects external stimulus information with feedback output behavior.In view of this,we intended to construct a subacute PD mouse model using MPTP,to observe and analyze the extent of dopamine neuron damage in the striatum and ventral midbrain and the changes in the behavioral phenotype of mice under different doses of treatment.At the same time,combined with chemical inheritance technology,we observed the effect of VTA neuron activation on anxiety behavior in mice.Objective:The behavioral phenotype of MPTP-induced subacute injury mice and the injury severity of DA neurons in striatum and ventral midbrain were observed in vivo.To clarify whether disturbing the balance of neurons in the residual VTA region on the basis of the MPTP-induced subacute PD mouse model induces the anxiety phenotype in mice,so as to establish a stable PD prodromal mouse model with anxiety state and clarify whether the neurons in the VTA region in the PD prodromal stage with anxiety participate in the neural circuit in anxiety state.Method:Two-month-old SPF grade C57BL/6J mice weighing 20–25 g were used to establish a subacute PD model by intraperitoneal injection of MPTP,and the AAV-9-hSyn-HA-hM3Dq-IRES-mCitrine virus vector was injected into the VTA site using brain stereotaxic technique.The behavioral experiments of full-automatic intelligent analysis cage,open field,stick rotation,gait,elevated cross maze,tail suspension and forced swimming of small animals were used to test the movement of mice and the anxiety and depression-related emotional disorders.Western blot was used to detect the protein expressions of striatal and ventral midbrain dopamine transporter(DAT),α-synuclein(α-Syn),tyrosine hydroxylase(TH),mixed lineage kinase domain-like(MLKL),and NOD-like receptor protein 3(NLRP3)inflammasomes.The expression and distribution of DA neurons,glial fibrillary acidic protein(GFAP)and ionized calcium binding adapter molecule 1(Iba1)positive cells in the striatum and ventral midbrain were detected by immunofluorescence.After the selective activation of neurons in the VTA region by chemical genetic technology,the open field,tail suspension and elevated cross maze experiments were performed to detect the movement of mice and the anxiety-depression related emotional disorders.The coexpression of HA tag with DA neurons,Glu neurons and GABA neurons in VTA was detected by immunofluorescence staining.The expression and distribution of c-fos positive cells in VTA,nucleus accumbens(NAc),ventral hippocampus(vHPC),basolateral amygdala(BLA)and medial prefrontal cortex(mPFC)were detected by immunofluorescence.Results:Study Ⅰ: To study the behavioral phenotype of a mouse model of Parkinson’ s disease induced by MPTP1.MPTP(160 mg/kg)-induced subacute injury mice do not show significant dyskinesia,but only show slight gait abnormalities;2.MPTP(160 mg/kg)PD model mice induce substantia nigra and striatum damage and show significant neuroinflammatory response;3.MPTP(160 mg/kg)-induced subacute injury in mice shows obvious astrocyte hyperplasia and microglia activation in the ventral midbrain and striatum;4.MPTP(240 mg/kg)-induced subacute injury mice do not show significant dyskinesia and show abnormal gait;5.MPTP(240 mg/kg)-induced subacute injury mice do not show significant anxiety and depression;6.MPTP(240 mg/kg)PD model mice significantly induce substantia nigra and striatum damage;7.MPTP(240 mg/kg)-induced neuronal over-activation in the VTA region in subacute injury mice;Study Ⅱ: Exploring the regulatory effect of VTA neurons on anxiety behavior in mice based on chemical genetic technology1.Chemical genetic activation of VTA regional neurons in female and male mice significantly enhances the intrinsic defensive response of mice in stressed environments;2.Chemical genetic activation of VTA regional neurons in female and male mice induces anxiety-like phenotype in mice;3.Chemical inheritance activates the neurons in the VTA region to participate in the regulation of mouse anxiety-like phenotype;4.VTA-NAc DA neurons may participate in the anxiety-like phenotype of mice;5.VTA-vHPC DA neurons may participate in the anxiety-like phenotype of mice;6.VTA-BLA DA-ergic neurons may participate in the anxiety-like phenotype of mice;7.The VTA-mPFC neural circuit may be involved in the anxiety-like phenotype of mice.Conclusion:1.MPTP-induced subacute PD mice correspond to the prodromal state of clinically PD patients—massive loss of DA neurons in the ventral midbrain of the mice and diffuse proliferation of astrocytes,but without showing significant motor and emotional disorders;2.Activation of VTA neurons,especially DA neurons,can induce anxiety-like behaviors in mice;In MPTP-induced subacute injury mice,the anxiety phenotype was relieved due to partial loss of VTA DA neurons;3.Each neuron of VTA,NAc,vHPC,BLA and mPFC together constitute an anxiety-related cell cluster and participate in the regulation of anxiety-like behavior in mice. |
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