Effects Of Tissue Type Plasminogen Activator On Gabaergic Neurons In The Visual Cortical Plasticity In Vitro

Objective:As a common clinical disease of Ophthalmology, Amblyopia is caused by receiving abnormal visual experience in the critical period of visual cortical plasticity. The visual acuity of the patients was decreased without any obvious organic disease. Amblyopia of older children and adults is extremely difficult to rescure, and has a low cure rate, further more, there is not even a effective treatment so far. As the corrected visual acuity of the patients is much less than the normal people’s, the disease gives rise to a lot of difficulties in the patients’s work, study and life.As the generation,development and treatment of the disease has a close relationship with the critical period(CP) visual cortical plasticity, research for the termination mechanism of the CP visual cortical plasticity has become the focus of a breakthrough for seeking new ways treating adult amblyopia. Recently, the role of tPA in the termination and reactivation of the visual cortical plasticity has received increasing attention.As a kind of serine protease,tPA helps to reorganize the neural connections of visual cortex by degradating the ECM or the recognition molecule of cell,and adjusts the balance between the inhibitory and excitatory neural circuits from the binocular zone of visual cortex, thus participates the development of the visual cortical plasticity and reactivates the plasticity which had been suppressed. However,the detailed action mechanism of tPA is still unclearly.Nowadays,many studies had indicated that the mature of GABA ergic neural circuits plays an important role on the action of tPA in the visual cortical plasticity.Therefore,we try to treat the cultured visual cortical neurons with tPA and PAI-1 in vitro and insight into the relationship among tPA,the GABA neurons and the GABAergic neural circuits in the structural and functional levels,so we can explore the possible mechanisms of tPAs participation and reactivation in the development of synaptic plasticity of visual cortex.Method:1 To provide a good source of neurons for the next experiment,we constructed a cultural system of rat visual cortical neurons’ in vitro, and identified the neurons from the morphologic and electrophysiologic function level.2 To explore the possible mechanisms of tPA-s participation and reactivation in the development of synaptic plasticity of visual cortex,we tried to enhance the tPA function with tPA and inhibite the tPA function with PAI-1.tPA Activity Kit was used to detect the effect of tPA and PAI-1 treatment on neurons in vitro. And GAD67/MAP2 immunofluorescence were used to detect the density and ratio of GABAergic neurons, the patch-clamp was used to test the sIPSCs,mIPSCs and mEPSCs for observing the effect of tPA and PAI-1 on the inhibitory synaptic transmission of GABAergic neural circuits.Result:1 The phase-contrast microscope results showed that the cultured neurons were fruity,and had clearly and bright boundary of cell; The neurons were identified successfully by the immunofluorescence of MAP2;The results of patch-clamp showed that three types of action potential could be induced from the neurons-----the single spike type, the transient type, the sustained type; frequent and spontaneous action potentials,classical whole cell current curve and miniature inhibitory postsynaptic currents could be recorded by the patch-clamp on the neurons in the later cultured period.2 We enhanced the tPA function with tPA and inhibited the tPA function with PAI-1.the results of tPA activity kit indicated that the tPA function was enhanced successfully in the tPA treatment group(P<0.05) and inhibited successfully in the PAI-1 treatment group(P<0.05).double-label immunofluorescence of GAD67/MAP2 results showed that the neurons-s density was no obvious difference among the control,tPA treatment and PAI-1 treatment group (.P>0.05). The GAD67 positive cell density and the ratio of GAD67/MAP2 were increased significantly in the group of PAI-1 treatment(P<0.05).However, those were no significant change in the group of tPA treatment(P>0.05); the patch-clamp results indicated that the amplitude and frequency of sIPSC, mlPSCs and mEPSCs were increased notable in PAI-1 treatment group(P<0.05).However,the frequency of sIPSC was decreased significantly (P<0.05),and the amplitude of sIPSCs,the amplitude and frequency of mlPSCs and mEPSCs were no obvious defference in the group of tPA treatment (P>0.05).Conclusion:1 We built a stable, effective and lowcost culture system of rat visual cortical neurons, the growth and electrophysiological function were both in good conditions.2 TPA can inhibit the formation of GABA neuronal circuits, and reduce the inhibitory synaptic transmission,induce the structure and transmission of GABA inhibitory synaptic tend to be naive.That may be the possible mechanisms of the participation and activation of tPA on the development of synaptic plasticity on visual cortex.