PTPσ受体参与神经元周围网络调节视皮层可塑性终止的机制研究
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摘要
弱视是指在视觉发育敏感期，由于缺乏清晰视觉图象刺激，从而造成矫正视力低于正常的疾病。弱视发病率高，视功能低下给患者的工作、学习和生活带来诸多困难。成人弱视发病率为2.9%，是20-70岁人群单眼视力损害的首要原因。弱视的发展和治疗与视皮层可塑性密切相关。在可塑性关键期内，采取适当的治疗方法能使弱视患者的视功能恢复；当可塑性关键期终止后(8-12岁以后)，虽然异常的视觉环境不能引起弱视，但弱视导致的视功能损害无法恢复。因此对视皮层可塑性机理进行研究，将进一步认识弱视的发病机制，并为提高成年弱视治愈率和开发弱视治疗的药物奠定实验基础。
当视皮层可塑性关键期终止时，视皮层细胞外间质(Extracellular matrix, ECM)中的硫酸软骨素蛋白多糖(chondroitin sulphate proteoglycans, CSPGs)等分子逐渐聚集并包绕神经元胞体和树突形成神经元周围网络(perineuronal net, PNNs)，主要包绕于Parvalbumin (PV)阳性神经元周围。采用chABC酶降解成年大鼠视皮层中的CSPGs后可以成功恢复视皮层眼优势柱的移动，并降低了视皮层γ-氨基丁酸(γ-aminobutyric acid, GABA)能神经元的抑制作用。CSPGs有共同的核心蛋白，并由胶质细胞和神经元共同分泌，能够抑制轴突的生长和再生。本实验室前期研究也发现随着视觉发育，正常大鼠视皮层神经元GABA受体介导的抑制性突触传递增强。应用chABC酶(chondroitinase ABC)降解大鼠视皮层PNNs中的CSPGs，并结合脑片膜片钳全细胞记录率先发现：ChABC酶处理组大鼠视皮层Ⅳ层神经元PSCs各项指标无显著变化，说明ChABC酶通过降解CSPGs并不影响视皮层Ⅳ层突触传递特性，但特异性地影响了GABAA介导的抑制性突触传递。上述结果提示CSPGs对视皮层发育可塑性关键期终止的作用可能是GABA能神经环路成熟和发挥抑制效应的结构基础。虽然既往研究观察到CSPGs是视皮层可塑性终止的关键因素，但并没有进一步探讨CSPGs发挥抑制作用的分子机制。
最新研究表明：酪氨酸磷酸酶蛋白受体σ (protein tyrosine phosphatase σ， PTPσ)为CSPGs发挥抑制轴突生长作用的特异性受体。PTPσ是白细胞抗原家族成员，由类免疫球蛋白(Ig)和纤维结合素Ⅲ (FNⅢ)重复片段等构成。该受体广泛分布于腺体和神经系统(高表达于海马，大脑皮质，嗅球，视网膜，室管膜下层)，参与了神经的发育及再生：主要表现为抑制轴突生长和参与胶质瘢痕的形成。PTPσ具有保守、正电荷胞外结构域，研究表明CSPGs中带负电荷的多糖链—硫酸软骨素(CS)正是PTPσ的结合位点。与ChABC酶降解CSPGs后的效果类似，体外实验发现即使CSPGs存在，功能性地删除编码PTPσ的基因Ptprs后，神经轴突仍然能够继续生长。在体实验也证实：Ptprs基因敲除动物脊髓背根节神经元轴突能够穿过脊髓损伤后CSPGs密集的瘢痕区域并继续生长。
之前的大量研究已经证实了PTPσ能够负向性地调节轴突的生长，但PTPσ受体的下游底物或胞内信号通路如何抑制轴突的生长和树突的形成，目前并不十分清楚。既往研究表明，肌动蛋白(actin)和视皮层可塑性的分子机制关系十分密切。神经元的细胞骨架包括微丝、微管及神经丝，这三种成分中只有含肌动蛋白网络的微丝在发育神经系统的生长锥和成熟神经系统的树突棘中发现。生长锥是个活跃运动的微观结构，其与神经突起的发生和生长、轴突的特殊途径选择，以及突触的形成有关，且神经突起的生长、延伸只发生在生长锥。研究发现作为突触局部细胞黏附分子(Synapticallylocalized cell adhesion molecules, SAMs)的成员，N-cadherin存在于突触前后末端，其胞内结构域和β-catenin结合，而后者则与α-catenin结合，从而使N-cadherin和actin形成连接。通过控制突触蛋白的聚集和树突棘的形态，N-cadherin和catenin蛋白参与了突触连接成熟的过程。Siu等研究发现，背根神经节神经元高度表达内源性的N-cadherin和PTPσ，且能够调控神经元生长锥的延伸。
结合上述内容我们提出假设：PTPσ参与了视皮层可塑性的终止，其机制与介导CSPGs对PV阳性神经元的调控作用有关，而PTPσ及其下游通路分子N-cadherin和β-catenin的表达变化是成年大鼠视皮层可塑性再激活的重要分子机制。
为此，本课题采用以下技术和方法，在大鼠视觉可塑性关键期及其终止前后，以及成年可塑性再激活状态下，探讨视皮层PTPσ与视觉发育可塑性关键期终止的关系及其可能存在的分子机制。
方法
1、应用荧光定量PCR观察PTPσ mRNA水平在发育前后的变化，以及免疫荧光组织化学研究PTPσ受体在视皮层中的表达和分布随发育的变化情况
2、应用免疫组织化学技术分别双重标记PTPσ和PNNs、PTPσ和PV阳性神经元，同时采用免疫荧光组织化学三重标记PV、PNNs、PTPσ，研究视皮层可塑性关键期的终止前后，PTPσ分别与PNNs、PV神经元在视皮层的共表达情况与发育之间的关系。
3、建立双眼形觉剥夺模型再激活成年大鼠视皮层可塑性后，采用免疫荧光组织化学观察PTPσ、PV、PNNs的表达情况。同时应用荧光定量PCR分别观察PTPσ、N-cadherin和β-catenin mRNA水平随发育以及在成年视皮层可塑性激活后的变化情况。
结果
1、PTPσ受体在视皮层可塑性关键期前后的表达和分布
(1)在大鼠出生后1周，PTPσ表达位于高水平，但在出生后3周以后，各层均显著下降，维持在低水平，说明PTPσ的表达呈现一定的视觉经验依赖性。
(2) PTPσ表达在可塑性关键期结束后，到成年期又升高恢复至可塑性开始时的水平，这为PTPσ参与视皮层可塑性关键期的“终止”提供了证据。
(3) PTPσ在其阳性表达细胞内表达定位于胞膜、胞浆及轴突。视皮层Ⅱ-Ⅲ层、Ⅳ层、Ⅴ-Ⅵ层均可见PTPσ阳性细胞表达，但各组均以Ⅴ-Ⅵ层表达最多。而视皮层中PTPσ阳性细胞绝大部分为神经元。
2、PTPσ受体调控Parvalbumin阳性神经元终止视皮层可塑性的研究
(1)绝大部分PNN阳性细胞表达PTPσ，提示视皮层中CSPGs可能通过PTPσ受体调节神经元的发育及成熟，并发挥抑制轴突生长的效应，是可塑性终止的重要分子机制。
(2)大部分PV阳性神经元表达PTPσ，提示在视皮层发育过程中PTPσ可能参与了PV神经元功能的调控，并一直持续到可塑性终止后至成年。同时，PV/PTPσ双标细胞在Ⅳ层最多，而在其他层较少，这与PNN/PTPσ双标细胞以及PNNs的分布特点相一致。
(3)视皮层中PV和PTPσ都为阳性表达的神经元也同时被PNN包绕，这一现象为PTPσ受体与CSPGs结合后发挥抑制PV神经元轴突生长及形成突触联系的效应提供了形态学证据。而视皮层IV层PV/PNN/PTPσ三标阳性神经元在PV神经元中的比例至PW9时最高，说明在视皮层发育过程中PTPσ逐渐参与了CSPGs对PV神经元的调控，可能是视皮层可塑性关键期终止的重要机制。
3、PTPσ受体及其下游通路分子参与成年大鼠视皮层可塑性再激活及其机制的研究
(1)双眼形觉剥夺后的大鼠与同龄成年大鼠相比，视皮层PTPσ mRNA明显下降，PTPσ阳性细胞密度在视皮层各层均明显减少，说明双眼形觉剥夺14天后降低了视皮层内PTPσ的表达水平。
(2)双眼形觉剥夺14天的大鼠与同龄成年大鼠相比，视皮层各层中PV神经元的数量并无明显变化。
(3)双眼形觉剥夺14天大鼠与同龄成年大鼠相比，视皮层与可塑性关系密切的Ⅱ-Ⅲ层、Ⅳ层CSPGs水平明显下降，说明成年大鼠视皮层可塑性再激活与CSPGs有关。
(4)视皮层N-cadherin mRNA的表达在PW1时最高随后下降，在PW7(可塑性终止前)再次出现高峰，并在成年后降至低水平，说明视皮层内N-cadherin mRNA的表达受到年龄及视觉经验等因素的影响，与可塑性终止有关。但双眼形觉剥夺14天对视皮层N-cadherin mRNA表达与同龄成年大鼠相比无明显变化。
(5)视皮层β-catenin mRNA的表达在PW1时最高随后下降，在PW7(可塑性终止前)再次出现高峰，并在成年后降至低水平，说明视皮层内β-catenin mRNA的表达受到年龄及视觉经验等因素的影响，与可塑性终止有关。然而，与同龄成年大鼠相比，双眼形觉剥夺14天能够使视皮层β-catenin mRNA表达升高。
结论
1.本实验首次揭示了出生后视皮层PTPσ受体的表达在可塑性关键期前后的发育变化，发现： PTPσ在出生后早期表达位于高水平，但在进入可塑性关键期后显著下降，维持在低水平，但当可塑性结束后，到成年期又升高恢复至可塑性开始时的水平。首次揭示PTPσ受体参与了视皮层可塑性关键期的“终止”。
2.在视皮层发育过程中大部分PV阳性神经元表达PTPσ，并一直持续到可塑性终止后至成年，提示在视皮层发育过程中PTPσ可能参与了PV神经元功能的调控。率先在组织水平上发现：视皮层中PV和PTPσ都为阳性表达的神经元也同时被PNN包绕，而视皮层IV层PV/PNN/PTPσ三标阳性神经元在PV神经元中的比例由关键期开始的最低值上升至成年期时的最高值，提示在视皮层发育过程中PTPσ逐渐参与了CSPGs对PV神经元的调控，是视皮层可塑性关键期终止的重要机制。
3.本实验率先发现双眼形觉剥夺14天使成年大鼠视皮层PTPσ、CSPGs表达明显下降，提示双眼形觉剥夺导致细胞外基质中的CSPGs及其受体PTPσ的减少，削弱了CSPGs的抑制效应，可能是成年大鼠视皮层可塑性“再激活”的机制之一。
4.视皮层β-catenin mRNA的表达在出生后早期最高随后下降，在可塑性终止前再次出现高峰，于成年后降至低水平，提示视皮层内β-catenin mRNA的表达受到年龄及视觉经验等因素的影响，与可塑性终止相关。率先发现双眼形觉剥夺能够使成年大鼠视皮层β-catenin mRNA表达升高，有利于促进了神经元突起生长，是成年大鼠可塑性“再激活”的机制之一。
Amblyopia is a common disease which can not be correceted to normal level andcaused by blurring visual pattern stimulation during the sensitive period of visualdevelopment. With high incidence, the lower visual function causes many difficulties in thejob, study and life of the amblyopia patients. The disease incidence in adults is2.9%, as thechief reason of the monocular impaired vison among people aging from20-70. Thedevelopment and treatment of this disease have closly related to the visual cortex plasticity.Within the critical period of plasticity, the visual function of ambyopia patients can berestored by some suitable methods. However, after the termination of critical period(usually after8-12years old), although abnormal visual enviroment can not causeamblyopia, but the impaired visual function can not recover. Thus it is very important tomake research on the mechanism of visual plasticity and know more about the pathogenesisof amblyopia, improving the cure rate of adult amblyopia patients and explore new drugsfor treating this disease.
Around the end of the critical period, the CSPGs in the ECM are gradually wrappingthe somata and dentrites of neurons, forming PNNs, especially around parvalbumin neurons.Degradation of CSPGs by the injection of chABCase can succesfully restore the movementof ocular dominance, by decreasing the inhitory function of GABA neurons. CSPGs havethe common core proteins and are secreted by glia cells and neurons, which could inhibitthe growth of axon and regeneration. Our previous work shows that the synapictransmission mediated by GABA receptors increased along with the development of vision.Degradation of CSPGs by ChaseABC in the rat visual cortex, and combined with the patchclamp technology, we found that the index of the PSCs did not change in layer Ⅳ,indicating that the degradation of CSPGs could not influence the characteristics of synaptic transmission in layer Ⅳ, but could affect the inhibitory synaptic transmissioin specifically.These results implied that the inhibition function of CSPGs in the termination of the criticalperiod might be the structural basis of the maturation of GABA neuronal circuits. Althoughit is noticed that CSPGs are the crucial factors in the termination of the visual cortex, theunderlying molecular mechanisms of CSPGs playing its inhibitory role are not clear.
Protein tyrosine phosphatase σ (PTPσ) is a member of LAR family, and comprisedof Ig and FNⅢ repeats, widely expressed in the glands and nervous system (highlyexpressed in hippocampus, cerebrum, bulbus olfactorius, retina and subependymal layer)participating in the process of neural development and regeneration, especially in theinhibition of the growth of the axon and the formation of the glia scar. PTPσ binds withhigh affinity to neural CSPGs, binding involves the chondroitin sulfate chains and a specificsite on the first immunoglobulin-like domain of PTPσ. Similarly to ChaseABC treatment,functional ablation of Ptprs, the gene encoding RPTPσ, promotes neurite outgrowth in thepresence of CSPGs in vitro and enhances axonal growth into CSPG-rich scar tissuefollowing SCI in vivo.
Recent research shows that PTPσ plays the role to negatively modulate the axonalgrowth as the functional receptor of the inhibitory function of CSPGs. However, it is stillunknown that whether PTPσis involved in the termination of critical period of visualcortex plasticity, as well as the correlation of CSPGs and PV neurons. Actin is closelyrelated to the molecular mechanism of the visual plasticity. The cytoskeleton of the neuronare microfilament, microtubule and neurofilament, only microfilament in the action network could befound in the developing growth cone and the spines in mature central nervous system. Growthcone is aactive movement microstructures, and participate in the genesis and growth of the nervous process,special pathway of axon, and formation of the synapses. Importantly, the growth and elongation of theneuritis can only be found in the growth cone. As a family member of the synaptically localized celladhesion molecules (SAMs), N-cadherin exists at the presynptic and postsynaptic terminals,and combined with β-catenin through the cellular structural domain. β-catenin combinedwith α-catenin, thus making the connection between N-cadherin and actin. Throughcontrolling the aggregation of synaptic proteins and the morphous of dentritic spines,N-cadherin and catenins participate in the process of the maturation of synapses. Siu alsofound that N-cadherin and PTPσ are highly expressed in dorsal root ganglion cells, and modulate the elongation of the neurites.
Besides, no report has been published on the changes of the downstream molecularsN-cadherin andβ-catenin during development and the reactivation of plasticity in adults.
Therefore, we applied the following methods and techniques to investigate therelationship between PTP σ and the termination of the critical period of visualdevelopment, as well as the possible underlying molecular mechanisms.
Methods:
1. With the use of Q-PCR and immunofluorescence histochemistry, we observed thePTPσ mRNA levels and the expression of PTPσ in the visual cortex during devlopment..2. With the use of immunofluorescence histochemistry to double labell PTPσ and PNNs,PTPσ and PV interneuron, triple immunostaining PTPσ, PNNs and PV neurons, wedetected the expression of PV, PNNs, PTPσ and their relationship during the developmentand the critical period.
3. By forming the binocular formation deprivation animal model to reactivating thevisual cortex plasticity in adult rats and being tested by P-VEPs to confirm the reactivationof the visual plasticity in the adult visual cortex, with the use of Q-PCR andimmunohistochemistry, we detected the change of PTPσ, PNNs and PV expression in alllayers of the visual cortex and also tested the change of N-cadherin and β-catenin mRNAlevels during development and after BFD in adult rats.
Results:
1. The expression and distribution of PTPσduring the development and the criticalperiod
(1) At PW1, the expression of PTPσ was at the highest level and then decreasedobviously after PW3in all layers, and stayed at a relatively low level during the criticalperiod, indicating that the expression of PTPσ is related to visual experience.
(2) The expression of PTPσ in adulthood increased to the same level of that at thebeginning of the critical period, giving proof that PTPσmay participate in the terminationof critical period.
(3) Cellular PTPσ was expressed in all layers of the VC through development. PTPσimmunolabeling appeared to be mainly localized within the cytoplasm and apical orprimary dendrites. PTPσ positive cells were found in all layers of visual cortex, which is mostly abundant in layer Ⅴ-Ⅵ. Interestingly, most PTPσ positive cells were neurons inthe visual cortex.
2. Research on PTPσmodulating PV neurons to terminate the visual cortical plasticity
(1) Most of the neurons wrapped by PNNs expressed PTPσ, indicating that CSPGsmay play its inhibitory role through its receptor PTPσ by inhibiting the axonal growth andmodulating the development and maturaion of PV neurons, which could be an importantmolecular mechanism of the termination of visual plasticity.
(2) Most of the PV neurons expressed PTPσ during the development, which suggestedthat PTPσ may be involved in the modulation of PV function until the adulthood. PV/PTPσdouble labelled cells were more intense in layer Ⅳ compared to other layers, whish was inconsisitence with the location feature of PNNs and PNN/PTPσ double immunostainingcells.
(3) The neurons expressed PTPσ and PV were also wrapped by PNNs, givingmorphological proof that CSPGs inhibit the axonal growth and the contact of synapses ofPV neurons by combining with PTPσ. The ratio of PV/PNN/PTPσ triple immunostainingneurons in PV neurons increased from the lowest ratio at PW3to the highest percentage atPW9, indicating that PTPσ gradually participate in the CSPG modulation of the PV neuronfunction, which was the important mechanism of the termination of visual plasticity.
3. Research on the participation of PTPσ and its downstream molecules in therestoration of plasticity in adult visual cortex
(1) Compared with the control rats (PW9, PTPσ mRNA level and the density of PTPσpositive cells in all layers were decreased in BFD group, suggesting that binocular formdeprivation could decrease the expression of PTPσ in the visual cortex of the adult rats.
(2) Binocular form deprivation did not change the number of PV neurons in all thelayers of the visual cortex.
(3) Binocular form deprivation could change the number of PNN postive cells in layerⅡ-Ⅲ and layer Ⅳ which are closly related to the visual cortical plasticity.
(4) The expression of N-cadherin mRNA was highest at PW1, then decreased until thelower peak at PW7, followed by the lower level in adulthood, suggesting that theexpression of N-cadherin mRNA was infuluenced by the development and visualexperience with related to plasticity. However, BFD didn’t change the N-cadherin mRNA level in the visual cortex.
(5) The expression of β-catenin mRNA was highest at PW1, then decreased until thelower peak at PW7, followed by the lower level in adulthood, suggesting that theexpression of β-catenin mRNA was infuluenced by the development and visual experiencewith related to plasticity. BFD increased the level of β-catenin mRNA to promote thegrowth of neurites, which may serve as the mechanism of the restoration of adult visualcortex plasticity.
Conclusion:
1. Our results found the expression of PTPσ during the development in the visualcortex for the first time. The expression of PTPσ was declined after PW1, and maintained atthe lowest level during the critical period. Interestingly, after the critical period (PW9), theexpression of PTPσ recovered to the same level at the start of the critical period (PW3).These results indicates that PTPσ is involved in the termination of the visual cortexplasticity.
2. Most of the PV neurons expressed PTPσ, which countinued to the end of the criticalperiod, even in the adulthood, indicating that PTPσ could modulate the function of PVneurons during the development of the visual cortex. The neurons expressed PTPσ and PVwere also wrapped by PNNs, and its ratio in PV neurons increased from the lowest at PW3(the beginning of the critical period) to the highest ratio at PW9(the adulthood), indicatingthat PTPσ participates in the modulation of the termination of visual cortex plasticity byparticipating in the CSPG modulation of the function of PV neurons.
3. We found that BFD14d could modulate the expression and distribution of CSPGsand PTPσ, suggesting that binocular form deprivation could decrease the expression ofCSPGs in the extracellular matrix and PTPσ, weaken the effect of the CSPG inhibition,could be one of the mechanism of the reactivation of the visual plasticity in the visualcortex of the adult rats.
4. The expression of β-catenin mRNA was found highest at PW1, then decreased withage. At the end of the critical period (PW7), it increased to the second peak, but with asignificant decrease in the adulthood (PW9), indicating that the expression of β-cateninmRNA could be modulated by factors of age and visual experience which are related to thetermination of visual cortex plasticity. However, binocular form deprivation could significantly increase the level of β-catenin mRNA, which could faciliate the growth of theneurite and might be an important mechanism of the restoration of visual cortex plasticityin adult rats.
当视皮层可塑性关键期终止时，视皮层细胞外间质(Extracellular matrix, ECM)中的硫酸软骨素蛋白多糖(chondroitin sulphate proteoglycans, CSPGs)等分子逐渐聚集并包绕神经元胞体和树突形成神经元周围网络(perineuronal net, PNNs)，主要包绕于Parvalbumin (PV)阳性神经元周围。采用chABC酶降解成年大鼠视皮层中的CSPGs后可以成功恢复视皮层眼优势柱的移动，并降低了视皮层γ-氨基丁酸(γ-aminobutyric acid, GABA)能神经元的抑制作用。CSPGs有共同的核心蛋白，并由胶质细胞和神经元共同分泌，能够抑制轴突的生长和再生。本实验室前期研究也发现随着视觉发育，正常大鼠视皮层神经元GABA受体介导的抑制性突触传递增强。应用chABC酶(chondroitinase ABC)降解大鼠视皮层PNNs中的CSPGs，并结合脑片膜片钳全细胞记录率先发现：ChABC酶处理组大鼠视皮层Ⅳ层神经元PSCs各项指标无显著变化，说明ChABC酶通过降解CSPGs并不影响视皮层Ⅳ层突触传递特性，但特异性地影响了GABAA介导的抑制性突触传递。上述结果提示CSPGs对视皮层发育可塑性关键期终止的作用可能是GABA能神经环路成熟和发挥抑制效应的结构基础。虽然既往研究观察到CSPGs是视皮层可塑性终止的关键因素，但并没有进一步探讨CSPGs发挥抑制作用的分子机制。
最新研究表明：酪氨酸磷酸酶蛋白受体σ (protein tyrosine phosphatase σ， PTPσ)为CSPGs发挥抑制轴突生长作用的特异性受体。PTPσ是白细胞抗原家族成员，由类免疫球蛋白(Ig)和纤维结合素Ⅲ (FNⅢ)重复片段等构成。该受体广泛分布于腺体和神经系统(高表达于海马，大脑皮质，嗅球，视网膜，室管膜下层)，参与了神经的发育及再生：主要表现为抑制轴突生长和参与胶质瘢痕的形成。PTPσ具有保守、正电荷胞外结构域，研究表明CSPGs中带负电荷的多糖链—硫酸软骨素(CS)正是PTPσ的结合位点。与ChABC酶降解CSPGs后的效果类似，体外实验发现即使CSPGs存在，功能性地删除编码PTPσ的基因Ptprs后，神经轴突仍然能够继续生长。在体实验也证实：Ptprs基因敲除动物脊髓背根节神经元轴突能够穿过脊髓损伤后CSPGs密集的瘢痕区域并继续生长。
之前的大量研究已经证实了PTPσ能够负向性地调节轴突的生长，但PTPσ受体的下游底物或胞内信号通路如何抑制轴突的生长和树突的形成，目前并不十分清楚。既往研究表明，肌动蛋白(actin)和视皮层可塑性的分子机制关系十分密切。神经元的细胞骨架包括微丝、微管及神经丝，这三种成分中只有含肌动蛋白网络的微丝在发育神经系统的生长锥和成熟神经系统的树突棘中发现。生长锥是个活跃运动的微观结构，其与神经突起的发生和生长、轴突的特殊途径选择，以及突触的形成有关，且神经突起的生长、延伸只发生在生长锥。研究发现作为突触局部细胞黏附分子(Synapticallylocalized cell adhesion molecules, SAMs)的成员，N-cadherin存在于突触前后末端，其胞内结构域和β-catenin结合，而后者则与α-catenin结合，从而使N-cadherin和actin形成连接。通过控制突触蛋白的聚集和树突棘的形态，N-cadherin和catenin蛋白参与了突触连接成熟的过程。Siu等研究发现，背根神经节神经元高度表达内源性的N-cadherin和PTPσ，且能够调控神经元生长锥的延伸。
结合上述内容我们提出假设：PTPσ参与了视皮层可塑性的终止，其机制与介导CSPGs对PV阳性神经元的调控作用有关，而PTPσ及其下游通路分子N-cadherin和β-catenin的表达变化是成年大鼠视皮层可塑性再激活的重要分子机制。
为此，本课题采用以下技术和方法，在大鼠视觉可塑性关键期及其终止前后，以及成年可塑性再激活状态下，探讨视皮层PTPσ与视觉发育可塑性关键期终止的关系及其可能存在的分子机制。
方法
1、应用荧光定量PCR观察PTPσ mRNA水平在发育前后的变化，以及免疫荧光组织化学研究PTPσ受体在视皮层中的表达和分布随发育的变化情况
2、应用免疫组织化学技术分别双重标记PTPσ和PNNs、PTPσ和PV阳性神经元，同时采用免疫荧光组织化学三重标记PV、PNNs、PTPσ，研究视皮层可塑性关键期的终止前后，PTPσ分别与PNNs、PV神经元在视皮层的共表达情况与发育之间的关系。
3、建立双眼形觉剥夺模型再激活成年大鼠视皮层可塑性后，采用免疫荧光组织化学观察PTPσ、PV、PNNs的表达情况。同时应用荧光定量PCR分别观察PTPσ、N-cadherin和β-catenin mRNA水平随发育以及在成年视皮层可塑性激活后的变化情况。
结果
1、PTPσ受体在视皮层可塑性关键期前后的表达和分布
(1)在大鼠出生后1周，PTPσ表达位于高水平，但在出生后3周以后，各层均显著下降，维持在低水平，说明PTPσ的表达呈现一定的视觉经验依赖性。
(2) PTPσ表达在可塑性关键期结束后，到成年期又升高恢复至可塑性开始时的水平，这为PTPσ参与视皮层可塑性关键期的“终止”提供了证据。
(3) PTPσ在其阳性表达细胞内表达定位于胞膜、胞浆及轴突。视皮层Ⅱ-Ⅲ层、Ⅳ层、Ⅴ-Ⅵ层均可见PTPσ阳性细胞表达，但各组均以Ⅴ-Ⅵ层表达最多。而视皮层中PTPσ阳性细胞绝大部分为神经元。
2、PTPσ受体调控Parvalbumin阳性神经元终止视皮层可塑性的研究
(1)绝大部分PNN阳性细胞表达PTPσ，提示视皮层中CSPGs可能通过PTPσ受体调节神经元的发育及成熟，并发挥抑制轴突生长的效应，是可塑性终止的重要分子机制。
(2)大部分PV阳性神经元表达PTPσ，提示在视皮层发育过程中PTPσ可能参与了PV神经元功能的调控，并一直持续到可塑性终止后至成年。同时，PV/PTPσ双标细胞在Ⅳ层最多，而在其他层较少，这与PNN/PTPσ双标细胞以及PNNs的分布特点相一致。
(3)视皮层中PV和PTPσ都为阳性表达的神经元也同时被PNN包绕，这一现象为PTPσ受体与CSPGs结合后发挥抑制PV神经元轴突生长及形成突触联系的效应提供了形态学证据。而视皮层IV层PV/PNN/PTPσ三标阳性神经元在PV神经元中的比例至PW9时最高，说明在视皮层发育过程中PTPσ逐渐参与了CSPGs对PV神经元的调控，可能是视皮层可塑性关键期终止的重要机制。
3、PTPσ受体及其下游通路分子参与成年大鼠视皮层可塑性再激活及其机制的研究
(1)双眼形觉剥夺后的大鼠与同龄成年大鼠相比，视皮层PTPσ mRNA明显下降，PTPσ阳性细胞密度在视皮层各层均明显减少，说明双眼形觉剥夺14天后降低了视皮层内PTPσ的表达水平。
(2)双眼形觉剥夺14天的大鼠与同龄成年大鼠相比，视皮层各层中PV神经元的数量并无明显变化。
(3)双眼形觉剥夺14天大鼠与同龄成年大鼠相比，视皮层与可塑性关系密切的Ⅱ-Ⅲ层、Ⅳ层CSPGs水平明显下降，说明成年大鼠视皮层可塑性再激活与CSPGs有关。
(4)视皮层N-cadherin mRNA的表达在PW1时最高随后下降，在PW7(可塑性终止前)再次出现高峰，并在成年后降至低水平，说明视皮层内N-cadherin mRNA的表达受到年龄及视觉经验等因素的影响，与可塑性终止有关。但双眼形觉剥夺14天对视皮层N-cadherin mRNA表达与同龄成年大鼠相比无明显变化。
(5)视皮层β-catenin mRNA的表达在PW1时最高随后下降，在PW7(可塑性终止前)再次出现高峰，并在成年后降至低水平，说明视皮层内β-catenin mRNA的表达受到年龄及视觉经验等因素的影响，与可塑性终止有关。然而，与同龄成年大鼠相比，双眼形觉剥夺14天能够使视皮层β-catenin mRNA表达升高。
结论
1.本实验首次揭示了出生后视皮层PTPσ受体的表达在可塑性关键期前后的发育变化，发现： PTPσ在出生后早期表达位于高水平，但在进入可塑性关键期后显著下降，维持在低水平，但当可塑性结束后，到成年期又升高恢复至可塑性开始时的水平。首次揭示PTPσ受体参与了视皮层可塑性关键期的“终止”。
2.在视皮层发育过程中大部分PV阳性神经元表达PTPσ，并一直持续到可塑性终止后至成年，提示在视皮层发育过程中PTPσ可能参与了PV神经元功能的调控。率先在组织水平上发现：视皮层中PV和PTPσ都为阳性表达的神经元也同时被PNN包绕，而视皮层IV层PV/PNN/PTPσ三标阳性神经元在PV神经元中的比例由关键期开始的最低值上升至成年期时的最高值，提示在视皮层发育过程中PTPσ逐渐参与了CSPGs对PV神经元的调控，是视皮层可塑性关键期终止的重要机制。
3.本实验率先发现双眼形觉剥夺14天使成年大鼠视皮层PTPσ、CSPGs表达明显下降，提示双眼形觉剥夺导致细胞外基质中的CSPGs及其受体PTPσ的减少，削弱了CSPGs的抑制效应，可能是成年大鼠视皮层可塑性“再激活”的机制之一。
4.视皮层β-catenin mRNA的表达在出生后早期最高随后下降，在可塑性终止前再次出现高峰，于成年后降至低水平，提示视皮层内β-catenin mRNA的表达受到年龄及视觉经验等因素的影响，与可塑性终止相关。率先发现双眼形觉剥夺能够使成年大鼠视皮层β-catenin mRNA表达升高，有利于促进了神经元突起生长，是成年大鼠可塑性“再激活”的机制之一。
Amblyopia is a common disease which can not be correceted to normal level andcaused by blurring visual pattern stimulation during the sensitive period of visualdevelopment. With high incidence, the lower visual function causes many difficulties in thejob, study and life of the amblyopia patients. The disease incidence in adults is2.9%, as thechief reason of the monocular impaired vison among people aging from20-70. Thedevelopment and treatment of this disease have closly related to the visual cortex plasticity.Within the critical period of plasticity, the visual function of ambyopia patients can berestored by some suitable methods. However, after the termination of critical period(usually after8-12years old), although abnormal visual enviroment can not causeamblyopia, but the impaired visual function can not recover. Thus it is very important tomake research on the mechanism of visual plasticity and know more about the pathogenesisof amblyopia, improving the cure rate of adult amblyopia patients and explore new drugsfor treating this disease.
Around the end of the critical period, the CSPGs in the ECM are gradually wrappingthe somata and dentrites of neurons, forming PNNs, especially around parvalbumin neurons.Degradation of CSPGs by the injection of chABCase can succesfully restore the movementof ocular dominance, by decreasing the inhitory function of GABA neurons. CSPGs havethe common core proteins and are secreted by glia cells and neurons, which could inhibitthe growth of axon and regeneration. Our previous work shows that the synapictransmission mediated by GABA receptors increased along with the development of vision.Degradation of CSPGs by ChaseABC in the rat visual cortex, and combined with the patchclamp technology, we found that the index of the PSCs did not change in layer Ⅳ,indicating that the degradation of CSPGs could not influence the characteristics of synaptic transmission in layer Ⅳ, but could affect the inhibitory synaptic transmissioin specifically.These results implied that the inhibition function of CSPGs in the termination of the criticalperiod might be the structural basis of the maturation of GABA neuronal circuits. Althoughit is noticed that CSPGs are the crucial factors in the termination of the visual cortex, theunderlying molecular mechanisms of CSPGs playing its inhibitory role are not clear.
Protein tyrosine phosphatase σ (PTPσ) is a member of LAR family, and comprisedof Ig and FNⅢ repeats, widely expressed in the glands and nervous system (highlyexpressed in hippocampus, cerebrum, bulbus olfactorius, retina and subependymal layer)participating in the process of neural development and regeneration, especially in theinhibition of the growth of the axon and the formation of the glia scar. PTPσ binds withhigh affinity to neural CSPGs, binding involves the chondroitin sulfate chains and a specificsite on the first immunoglobulin-like domain of PTPσ. Similarly to ChaseABC treatment,functional ablation of Ptprs, the gene encoding RPTPσ, promotes neurite outgrowth in thepresence of CSPGs in vitro and enhances axonal growth into CSPG-rich scar tissuefollowing SCI in vivo.
Recent research shows that PTPσ plays the role to negatively modulate the axonalgrowth as the functional receptor of the inhibitory function of CSPGs. However, it is stillunknown that whether PTPσis involved in the termination of critical period of visualcortex plasticity, as well as the correlation of CSPGs and PV neurons. Actin is closelyrelated to the molecular mechanism of the visual plasticity. The cytoskeleton of the neuronare microfilament, microtubule and neurofilament, only microfilament in the action network could befound in the developing growth cone and the spines in mature central nervous system. Growthcone is aactive movement microstructures, and participate in the genesis and growth of the nervous process,special pathway of axon, and formation of the synapses. Importantly, the growth and elongation of theneuritis can only be found in the growth cone. As a family member of the synaptically localized celladhesion molecules (SAMs), N-cadherin exists at the presynptic and postsynaptic terminals,and combined with β-catenin through the cellular structural domain. β-catenin combinedwith α-catenin, thus making the connection between N-cadherin and actin. Throughcontrolling the aggregation of synaptic proteins and the morphous of dentritic spines,N-cadherin and catenins participate in the process of the maturation of synapses. Siu alsofound that N-cadherin and PTPσ are highly expressed in dorsal root ganglion cells, and modulate the elongation of the neurites.
Besides, no report has been published on the changes of the downstream molecularsN-cadherin andβ-catenin during development and the reactivation of plasticity in adults.
Therefore, we applied the following methods and techniques to investigate therelationship between PTP σ and the termination of the critical period of visualdevelopment, as well as the possible underlying molecular mechanisms.
Methods:
1. With the use of Q-PCR and immunofluorescence histochemistry, we observed thePTPσ mRNA levels and the expression of PTPσ in the visual cortex during devlopment..2. With the use of immunofluorescence histochemistry to double labell PTPσ and PNNs,PTPσ and PV interneuron, triple immunostaining PTPσ, PNNs and PV neurons, wedetected the expression of PV, PNNs, PTPσ and their relationship during the developmentand the critical period.
3. By forming the binocular formation deprivation animal model to reactivating thevisual cortex plasticity in adult rats and being tested by P-VEPs to confirm the reactivationof the visual plasticity in the adult visual cortex, with the use of Q-PCR andimmunohistochemistry, we detected the change of PTPσ, PNNs and PV expression in alllayers of the visual cortex and also tested the change of N-cadherin and β-catenin mRNAlevels during development and after BFD in adult rats.
Results:
1. The expression and distribution of PTPσduring the development and the criticalperiod
(1) At PW1, the expression of PTPσ was at the highest level and then decreasedobviously after PW3in all layers, and stayed at a relatively low level during the criticalperiod, indicating that the expression of PTPσ is related to visual experience.
(2) The expression of PTPσ in adulthood increased to the same level of that at thebeginning of the critical period, giving proof that PTPσmay participate in the terminationof critical period.
(3) Cellular PTPσ was expressed in all layers of the VC through development. PTPσimmunolabeling appeared to be mainly localized within the cytoplasm and apical orprimary dendrites. PTPσ positive cells were found in all layers of visual cortex, which is mostly abundant in layer Ⅴ-Ⅵ. Interestingly, most PTPσ positive cells were neurons inthe visual cortex.
2. Research on PTPσmodulating PV neurons to terminate the visual cortical plasticity
(1) Most of the neurons wrapped by PNNs expressed PTPσ, indicating that CSPGsmay play its inhibitory role through its receptor PTPσ by inhibiting the axonal growth andmodulating the development and maturaion of PV neurons, which could be an importantmolecular mechanism of the termination of visual plasticity.
(2) Most of the PV neurons expressed PTPσ during the development, which suggestedthat PTPσ may be involved in the modulation of PV function until the adulthood. PV/PTPσdouble labelled cells were more intense in layer Ⅳ compared to other layers, whish was inconsisitence with the location feature of PNNs and PNN/PTPσ double immunostainingcells.
(3) The neurons expressed PTPσ and PV were also wrapped by PNNs, givingmorphological proof that CSPGs inhibit the axonal growth and the contact of synapses ofPV neurons by combining with PTPσ. The ratio of PV/PNN/PTPσ triple immunostainingneurons in PV neurons increased from the lowest ratio at PW3to the highest percentage atPW9, indicating that PTPσ gradually participate in the CSPG modulation of the PV neuronfunction, which was the important mechanism of the termination of visual plasticity.
3. Research on the participation of PTPσ and its downstream molecules in therestoration of plasticity in adult visual cortex
(1) Compared with the control rats (PW9, PTPσ mRNA level and the density of PTPσpositive cells in all layers were decreased in BFD group, suggesting that binocular formdeprivation could decrease the expression of PTPσ in the visual cortex of the adult rats.
(2) Binocular form deprivation did not change the number of PV neurons in all thelayers of the visual cortex.
(3) Binocular form deprivation could change the number of PNN postive cells in layerⅡ-Ⅲ and layer Ⅳ which are closly related to the visual cortical plasticity.
(4) The expression of N-cadherin mRNA was highest at PW1, then decreased until thelower peak at PW7, followed by the lower level in adulthood, suggesting that theexpression of N-cadherin mRNA was infuluenced by the development and visualexperience with related to plasticity. However, BFD didn’t change the N-cadherin mRNA level in the visual cortex.
(5) The expression of β-catenin mRNA was highest at PW1, then decreased until thelower peak at PW7, followed by the lower level in adulthood, suggesting that theexpression of β-catenin mRNA was infuluenced by the development and visual experiencewith related to plasticity. BFD increased the level of β-catenin mRNA to promote thegrowth of neurites, which may serve as the mechanism of the restoration of adult visualcortex plasticity.
Conclusion:
1. Our results found the expression of PTPσ during the development in the visualcortex for the first time. The expression of PTPσ was declined after PW1, and maintained atthe lowest level during the critical period. Interestingly, after the critical period (PW9), theexpression of PTPσ recovered to the same level at the start of the critical period (PW3).These results indicates that PTPσ is involved in the termination of the visual cortexplasticity.
2. Most of the PV neurons expressed PTPσ, which countinued to the end of the criticalperiod, even in the adulthood, indicating that PTPσ could modulate the function of PVneurons during the development of the visual cortex. The neurons expressed PTPσ and PVwere also wrapped by PNNs, and its ratio in PV neurons increased from the lowest at PW3(the beginning of the critical period) to the highest ratio at PW9(the adulthood), indicatingthat PTPσ participates in the modulation of the termination of visual cortex plasticity byparticipating in the CSPG modulation of the function of PV neurons.
3. We found that BFD14d could modulate the expression and distribution of CSPGsand PTPσ, suggesting that binocular form deprivation could decrease the expression ofCSPGs in the extracellular matrix and PTPσ, weaken the effect of the CSPG inhibition,could be one of the mechanism of the reactivation of the visual plasticity in the visualcortex of the adult rats.
4. The expression of β-catenin mRNA was found highest at PW1, then decreased withage. At the end of the critical period (PW7), it increased to the second peak, but with asignificant decrease in the adulthood (PW9), indicating that the expression of β-cateninmRNA could be modulated by factors of age and visual experience which are related to thetermination of visual cortex plasticity. However, binocular form deprivation could significantly increase the level of β-catenin mRNA, which could faciliate the growth of theneurite and might be an important mechanism of the restoration of visual cortex plasticityin adult rats.
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