Effect Of CSPG On Neuronal Excitability Circuit In Rat Visual Cortex Around The End Of Critical Period Of Visual Plasticity

Amblyopia is a common disease in ophthalmology. In world, 2-5% population are amblyopia patients including more than 10 million amblyopia children in China. Amblyopia patients have correct acuity lower than that of normal person at same age, impaired contrast sensitivity, stereoscopic vision and motion perception. Many studies suggest that visual cortex is the crucial impaired target of amblyopia. Development and therapy of amblyopia have relation with the critical period of visual plasticity. During critical period of visual plasticity, abnormal visual experience, such as congenital cataract and strabilismus, can lead to amblyopia and amblyopia can be cured after abnormal visual experience removed. After the end of critical period of visual plasticity, abnormal visual experience can not lead to amblyopia and amblyopia developed previously can not be cured. Clinically adult amblyopia patients and amblyopia children elder than 12 year can not be cured. Therefore whether the critical period of visual plasticity end or not has very important significance for therapeutic efficacy of amblyopia.Development of the visual cortex is strongly modified by visual experience during early short period of postnatal development called critical period of visual plasticity. Recently investigations suggest that factors have effects on the end of critical period of visual plasticity, including synaptic plasticity in visual cortex, maturation of excitatory circuit and inhibitory circuit, extracellular matrix, neurotrophic factors and expression of related gene. Recent studies found that chondroitin sulphate proteoglycan(CSPG), a component of extracellular matrix, condensed around a group of neurons and formed perineuronal nets(PNNs) encapsuling the soma and dendrites of neurons in central nervous system. The PNNs was formed with CSPG during the late phase of critical period. Dark rearing delay the end of critical period of visual plasticity, also decrease the number of PNNs, suggesting that the PNNs formed with CSPG is involved in the end of critical period of visual plasticity.In rat visual cortex, glutamatergic excitatory transmission is mediated by NMDA receptor and AMPA receptor. After the peak of critical period of rat visual plasticity, the proportion of NMDA receptor mediated excitatory postsynaptic current(NMDA-EPSC) in glutamatergic excitatory postsynaptic current(Glu-EPSC) gradually decrease and the proportion of AMPA receptor mediated excitatory postsynaptic current(AMPA-EPSC) in Glu-EPSC gradually increase. Dark rearing can delay developmental changes in properties of NMDA-EPSC in rat visual cortex, implying that developmental changes in Glu-EPSC might be involved in the end of critical period of visual plasticity. These investigations suggest that developmental changes in CSPG and excitatory synapses have relation with the end of critical period of visual plasticity. However mechanisms ending the critical period are not well known.We hypothesized that: During late phase of critical period, the PNNs formed with CSPG encapsule the soma and dendrites of neurons in visual cortex, and preferentially"shield"NMDA receptor. This leads to NMDA receptor mediated component in excitatory transmission decrease, and AMPA receptor mediated component in excitatory transmission increase, which result in the plasticity of excitatory synapses decrease gradually and the end of critical period of visual plasticity. To identify this hypothesis, the present study was performed.Part 1. Developmental changes in glutamatergic excitatory postsynaptic current in normal rat visual cortex around the end of cirtical period.Using patch clamp technique, we studied developmental changes in Glu-EPSC, NMDA-EPSC and AMPA-EPSC in normal rat visual cortex from P3W to P8W. Results in this study suggested that: 1. The NMDA-EPSC amplitude had no significant changes from P3W to P8W, whereas amplitudes of Glu-EPSC and AMPA-EPSC increased gradually from P3W to P8W, achieved peak value at P7W and P6W, respectively. 2. The NMDA-EPSC/Glu-EPSC ratio decreased gradually and achieved lowest level at P6W, whereas the AMPA-EPSC/Glu-EPSC ratio increased gradually and achieved peak level at P6W. These results indicated that developmental changes in NMDA-EPSC/Glu-EPSC ratio and AMPA-EPSC/Glu-EPSC ratio might be one of mechanisms ending the critical period of visual plasticity.Part 2. Effects of degradating CSPG on Glu-EPSC, NMDA-EPSC and AMPA-EPSC in rat visual cortexCSPG degradation rats were made by normal rats visual cortex chondroitinase ABC(chABC) injection. Developmental changes in Glu-EPSC, NMDA-EPSC and AMPA-EPSC in CSPG degradation rat visual cortex were studied by patch clamp technique. To explore the effect of CSPG on developmental changes in Glu-EPSC, NMDA-EPSC and AMPA-EPSC in rat visual cortex, data from CSPG degradation rats were compared with those from normal rats. Results in this study suggested that: 1. NMDA-EPSC amplitudes of CSPG degradation rats were significant higher than those of normal rats from P4W to P8W. AMPA-EPSC amplitudes of CSPG degradation rats were significant higher than those of normal rats from P6W to P8W. 2.From P6W to P8W, the NMDA-EPSC/Glu-EPSC ratio of CSPG degradation rat was significant higher than that of normal rat, whereas the AMPA-EPSC/Glu-EPSC ratio of CSPG degradation rat was significant lower than that of normal rat. 3. After CSPG degradated, the increase percentage of NMDA-EPSC amplitude was higher than that of AMPA-EPSC amplitude in rat visual cortex. These results suggested that CSPG preferentially had inhibitory effect on the NMDA-EPSC amplitude in rat visual cortex before the end of critical period.Part 3. Effects of degradating CSPG on expression of NMDA receptor and AMPA receptor in rat visual cortexUsing immunofluorescence, we investigated developmental changes in NMDA receptor subunit NR1 immunoreactive(NR1-IR) neurons and AMPA receptor subunit GluR2 immunoreactive(GluR2-IR) neurons in visual cortex of CSPG degradation rat and normal rat. Results in this study suggested that: 1. The number of NR1-IR neurons in normal rat visual cortex layer 2-3 had no significant changes from P3W to P8W. The number of NR1-IR neurons in normal rat visual cortex layer 4 decreased from P3W to P5W, and had no significant changes from P5W to P8W. The number of NR1-IR neurons in visual cortex layer 2-4 of CSPG degradation rat was significant higher than that of normal rat from P4W to P8W. 2. The number of GluR2-IR neurons in normal rat visual cortex layer 2-3 gradually increased from P3W to P8W,whereas the number of GluR2-IR neurons in normal rat visual cortex layer 4 gradually increased from P3W to P5W and had no significant changes from P5W to P8W. The number of GluR2-IR neurons in CSPG degradation rat visual cortex layer 2-3 was higher than that in normal rat visual cortex from P6W to P8W. The number of GluR2-IR neurons in CSPG degradation rat visual cortex layer 4 was higher than that in normal rat visual cortex from P7W to P8W. 3. After CSPG degradated, the increase percentage of NR1-IR neurons in rat visual cortex layer 2-4 was higher than that of GluR2-IR neurons. Using western blot, we investigated developmental changes in NR1 level and GluR2 level in visual cortex of CSPG degradation rat and normal rat. Results analyzed by western blot suggested that: 1. NR1 level in normal rat visual cortex decreased gradually. NR1 level in normal rat visual cortex from P3W to P4W were higher that those from P7W to P8W. NR1 level in CSPG degradation rat visual cortex was significant higher than that in normal rat visual cortex from P4W to P8W. 2. GluR2 level in normal rat visual cortex increased gradually from P3W to P6W and had no significant changes from P6W to P8W, whereas GluR2 level in CSPG degradaton rat visual cortex was higher than that in normal rat visual cortex from P6W to P8W.These results indicated that from the late phase to the end of critical period of visual plasticity, NMDA receptor had been present, whereas expression of NR1 protein was"shield"preferentially by PNNs formed with CSPG. This blocked the combination of transmitter and NMDA receptor. In addition, these results indicated that CSPG had only effects on AMPA receptor during adulthood.Summarized results in present study, it could be concluded that from the peak to the end of critical period, the PNNs formed with CSPG preferentially"shield"expression and function of NMDA receptor in rat visual cortex, leading to relatively enhancement in expression and function of AMPA receptor. This results in that AMPA receptor had predominate role in excitatory transmission around the end of critical period. These changes in NMDA receptor and AMPA receptor lead to that glutamatergic excitatory synapses were strengthened, synaptic plasticity decreased and critical period closured. Thus the PNNs formed with CSPG preferentially"shield"NMDA receptor in rat visual cortex lead to changes in constituent ratio of NMDA receptor and AMPA receptor in excitatory neuronal network in rat visual cortex that might be one of mechanisms ending critical period of visual plasticity.