Study On Mechanisms By Which TPA Terminate The Critical Period Of Rat Visual Development

Visual development, a branch of neuroscience, is an important field for clinical and theoretical research in ophthalmology. Investigation of visual development will help interpret the principle of brain working, and provide theoretical basis for the visual recostruction of adult visual damage and the treatment of infant-eye-disease.Amblyopia is a common disease in ophthalmology, and 2 to 5 percents of people all around the world are amblyopia patients including more than 10 million amblyopia children in China. Amblyopia is a disorder referring to impaired vision without obvious pathological changes in ophthalmic examinations, which is resulted from insufficient visual information received by visual neurons during the critical period of rat visual development. Amblyopia patients have not only lower monocular vision than normal ones, but also poor binocular vision, which make it difficult for them to work, study and live normally in daily life. However, there have no effective drug treatments for amblyopia until now. For the onset, development, and treatment of amblyopia are closely related with critical period of visual cortex plasticity, study on mechanisms terminating the critical period of visual development will help provide theoretical basis for improving curative effect for amblyopia and drug treatment research on it.Around the end of critical period of visual development, GABAergic inhibitory interneuron are embraced more and more by PNNs, which is composed of CSPGs moleculars in ECM of visual cortex and contributes to prevent the outgrowth of axons. Degrading PNNs in visual cortex of adult rat can reactivate ocular dominance plasticity, and decrease GABA inhibition to its target cells. The above investigations indicate that the maturation of GABAergic inhibitory circuits plays an important role in temination of critical period, and the formation of PNNs promotes the functional maturation of GABAergic inhibitory interneurons.Tissue-type plasminogen activator(tPA)is a major serine protease in postnatal mammal's central nervous system. tPA play a key role in mediating ocular dominance shift in response to monocular deprivation by acting on plasmin and forming the tPA/plasmin cascade activation system, which can directly or indirectly degrade a variety of extracellular moleculars. However, it is unkown whether tPA is involved in the termination of critical period of visual cortex plasticity, as well as the correlation of its intracellular mechanism and ECM moleculars. To date, no report has been published on this subject.Therefore, we applied the following methods and techniques to investigate the relationship between tPA and the end of critical period of visual development, as well as the possible inner mechanism.1. With the use of immunofluorescence histochemistry, immunoblot, and chromogenic assay kit, we detected tPA expression and activity in visual cortex during the postnatal development. The results show that (1) No tPA was found in visual cortex in the rats of 1 week group; (2) tPA was detected with the highest expression in the late phaseof critical period and the lowest expression in adult. The tPA activity was decreasing during postnatal development with the lowest level in adult. (3) From the peak, late phase, end of critical period to adult, tPA was positive in the all layers of visual cortex with major location in layerâ…¡-â…¢andâ…£. (4)In layerâ…¡-â…¢, the density of tPA-containing cells reached peak at late phase of critical period, and then reduced to minimum at adult. In the layerâ…£, the density of tPA-containing cells attached the maximum at peak of critical period and then decreased to the minimum at adult.2.With the use of immunofluorescence histochemistry to double labell tPA and PNNs,tPA and GABAergic interneuron,and immunobloting to detect GAD65(one of GABA synthetic limiting-speed enzyme) expression during development. The results show that (1)GABAergic interneuron and PNNs both remained increasing in development, and had a significant upregulation at late phase of critical period. tPA-positive neuron expression reached peak at late phase of critical period, and significantly decreased in later development.(2) From late phase of critical period to adult, tPA/PNNs,tPA/GABA double labeling neuron expression were both significantly downregulated, and declined to the low adult expression level at end of critical period, which is the same as tPA-positive neuron development.3. With the use of path-clamp whole-cell recording technique and immunohistochemistry to investigate the effect of exogenous regulation of tPA(given exogenous tPA or its inhibitor tPA-stop to infuse and incubate visual cortex slice) on PNNs and GABAA-IPSCs of layer IV neurons in visual cortex before and after the closure of critical period. The results show that:(1) exogenous tPA or its inhibitor tPA-stop infusion on visual cortex slice had no influence in number of PNNs, but exogenous tPA infusion degraded PNNs in its appearance.(2) exogenous tPA or its inhibitor infusion on visual cortex slice had no influence in PSCs and passive membrane characteristics.(3) exogenous tPA infusion made the decay time and peak value of GABAA-IPSCs significantly decrease, and exogenous tPA-stop infusion had no these effects.(4) exogenous tPA infusion made the developmental curves of decay time, peak value, IPSCs/PSCs ratio of GABAA-IPSCs move posterior,and exogenous tPA-stop infusion made them move forward.To sum up, we conclued as the following: 1. tPA is involved in the termination of visual cortex plasticity. 2. From the late phase of critical period(PW 5) on, both of the development of PNNs and GABAergic inhibitory neurotransmission system are nearly mature simultaneously, but tPA-positive expression is hardly downregulated with development. These suggest from the point of structure that tPA terminate the critical period of rat visual development may be due to its developmental downregulation which is permissive for PNNs formation and the latter maturation of GABA inhibitory neural circuit. 3. exogenous tPA has inhibitory effect on maturation of GABAA mediated inhibitory synaptic neurotransmission and thus contributes to delaying ctitical period of plasticity. Exogenous tPA,tPA-stop infusion can change the developmental properties of GABAergic inhibitory synaptic function in layer IV of visual cortex, resulting in regulation of termination of critical period. To conclusion, the mechanisms by which tPA terminate the critical period of rat visual development may be due to its developmental downregulation which is permissive for PNNs formation and the latter maturation of GABA inhibitory neural circuit advanced by PNNs.