cAMP反应元件结合蛋白(CREB)在正常发育大鼠视皮层的表达研究

正常视觉信息输入对初期视皮层的发育和修饰至关重要。视觉发育的特点是存在一个关键期 ,在此期内控制信息输入将导致皮质联系的显著变化。转录因子 -c AMP反应元件结合蛋白 ( CREB)在多种有机体的信号传导通路中的枢纽作用和其参与长时程突触可塑性的生理活动提示 ,它可能参与了在视皮层发育中发挥重要作用的分子机制。本研究应用免疫组织化学方法和 Western blot技术 ,对 P14、P2 2、P3 0、P45和 P90年龄组 Sprague-Dawley大鼠视皮层内 CREB的免疫反应性进行观察和分析。结果证明 ,CREB在视皮层各层内的蛋白表达时程与视觉发育的关键期一致 ,其免疫反应性在 P2 2至 P45期间达到高水平 ,成年后的蛋白表达出现下调。结论 :视觉可塑性降低的同时存在 CREB表达的显著改变 ,但该蛋白表达改变与突触传递和可塑性的关系尚待阐明     

细胞外信号调节激酶系统(ERKs)在正常发育和单眼剥夺大鼠视皮层蛋白表达的研究

为了研究正常发育和单眼视觉剥夺大鼠视皮层 ERK1/ ERK2基因蛋白质表达的变化 ,本研究建立了 SD大鼠单眼视觉剥夺模型 ,以多克隆抗 ERK1/ ERK2抗体和单克隆抗双磷酸化 ERK抗体检测出生后第 1、14、2 1、2 8、45 d和成年 (90 d)正常发育和单眼视觉剥夺 (14～ 45 d)视皮层 ERK1/ ERK2蛋白表达和活性改变。结果表明 ,出生后大鼠视皮层 ERK1/ ERK2蛋白表达逐渐增加 ,至 45 d达到高峰 ,此后下降至成年达稳定水平。磷酸化 ERK蛋白表达仅见于成年大鼠视皮层。单眼视觉剥夺导致ERK1/ ERK2蛋白表达减少。提示 ERK的蛋白表达依赖于正常的视觉输入信号的刺激 ,异常的视觉经验造成表达的明显下调 ,阻断正常的发育进程。说明 ERK1和 ERK2可能参与发育敏感期视皮层神经元可塑性的调节     

蛋白激酶α和β在视觉剥夺性大鼠视皮层中表达变化的初步研究

目的　检测蛋白激酶α和 β(PKCα、PKCβ)在正常大鼠视皮层及视觉剥夺性视皮层中的蛋白表达情况 ,为探讨视觉发育可塑性提供分子基础。方法　采用剥夺性弱视大鼠模型 ,用特异的PKCα和PKCβ抗体对脑切片进行免疫组化。 结果　正常视皮层中 ,这两种PKC同工酶在除Ⅰ层外的其余各层均有显著表达。视觉剥夺的视皮层缺乏正常视皮层那样的清晰分层。与正常视皮层比较 ,弱视眼对侧视皮层中PKCα在Ⅱ～Ⅳ层的蛋白表达广泛减少 ,PKCβ在Ⅱ～Ⅴ层的表达强度明显减弱。结论　PKCα、PKCβ的蛋白表达水平在剥夺性视皮层发育障碍过程中发生了显著改变 ,它们可能参与视皮层发育可塑性的分子机制。     

核呼吸因子-1在正常发育和视觉剥夺大鼠视皮层内的表达

目的:观察核呼吸因子-1(NRF-1)在正常发育及视觉剥夺大鼠视皮层内的表达.方法:构建视觉剥夺大鼠模型,利用半定量:PCR、Western blot及免疫组织化学的方法检测NRF-1在正常发育及视觉剥夺大鼠视皮层内的差异表达.结果:通过以上方法均可检测到视觉剥夺大鼠视皮层内NRF-1表达明显降低.结论:NRF-1的基因转录及蛋白表达水平依赖于正常视觉信号的刺激,异常的视觉经验使视皮层神经元兴奋性降低,造成NRF-1表达的明显下调.     

单侧和双侧眼睑缝合后大鼠脑内视觉系统星形胶质细胞的反应

目的　探讨单侧和双侧眼睑缝合后 ,幼年大鼠脑内视觉系统星形胶质细胞可塑性的变化。　方法　分别缝合出生后 7d大鼠单侧或双侧眼睑并维持 80d ,正常光线环境下饲养大鼠作为对照组。应用免疫组织化学ABC法观察大鼠脑内视觉系统胶质原纤维酸性蛋白的变化。　结果　与对照组相比单眼与双眼缝合组大鼠脑内GFAP阳性结构均减少。单侧眼睑缝合组视交叉、视束及缝合眼对侧与视觉传导相关的脑区 (包括视交叉上核、外侧膝状体、枕叶视皮质、上丘视神经层、顶盖前区 )内GFAP阳性结构明显减少。双侧枕叶视皮质呈现“互补分布”的特点。双侧眼睑缝合组大脑两侧上述脑区内GFAP阳性结构基本消失。单侧和双侧眼睑缝合组两侧嗅皮层内GFAP阳性结构明显增加。　结论　提示发育过程中视觉经验可以影响星形胶质细胞的结构     

PGC-1,NRF-2α和mtTFA在视觉剥夺大鼠视皮质内的表达下调

 目的 观察核呼吸因子-2α(NRF-2α)在正常发育及视觉剥夺大鼠视皮质内的表达。方法 构建视觉剥夺大鼠模型,利用半定量PCR和免疫印迹的方法检测过氧化物酶体增生激活受体γ协同刺激因子(PGC-1)、 NRF-2α和线粒体转录因子A(mtTFA)在视皮质内的表达。结果 视觉剥夺大鼠视皮质内PGC-1、NRF-2α和mtTFA表达较正常组明显降低(P < 0.05)。结论NRF-2α的基因转录及蛋白表达水平依赖于正常视觉信号的刺激,异常的视觉经验使视皮质神经元兴奋性降低,造成NRF-2α表达的明显下调。     

细胞外信号调节激酶系统 (ERKs)在正常发育大鼠视皮层基因表达的研究

细胞外信号调节蛋白激酶 (ERKs)是皮层神经元生长、发育和分化的关键因子。本研究目的在于研究 ERKs(ERK1、ERK2和 ERK3 ) m RNA在视皮层各层的分布、表达量以及发育过程变化。实验用健康雄性 SD大鼠 ,于生后 (P) 14、2 1、2 8、45和 90 d(成年 )灌注固定 ,取全脑 ,切取视皮层。用 4%多聚甲醛固定 ,石蜡包埋 ,4μm厚切片。地高辛标记特异性寡核苷酸探针 (ERK1、ERK2 )和 c DNA探针 (ERK3 )。用原位杂交方法检测三种 ERKs亚型的 m RNA在各年龄组大鼠视皮层的表达。结果证明 :ERKs m RNA在出生后大鼠正常发育视皮层的表达 ,ERK1和 ERK2 m RNA的分布具有明显的层的特异性 ,表达于除 I层 (分子层 )之外的 II-VI层 ,ERK2较 ERK1m RNA的表达更广泛、信号密度更强。ERK1和 ERK2 m RNA的转录在发育敏感期增高 ,从 P2 1～P2 8逐渐增加 ,P45时达到高峰 ,到成年时降低为相当于 P2 1的水平。 ERK3 m RNA在大鼠出生后视皮层的信号表达强 ,比较恒定 ,无明显的层分布特异性。本研究结果提示 ,出生后正常大鼠发育期视皮层 ERK1和 ERK2的 m RNA表达呈上调趋势 ,而 ERK3 m RNA在大鼠出生后视皮层的表达量中等 ,比较恒定 ,缺乏发育性变化特点。表明 ERK1和 ERK2可能是参与出生后在视觉环境刺激下视皮层发育可塑性调节的重要     

大鼠睁眼前初级视皮层2/3层锥体神经元突触自身稳态可塑性的变化特征

目的：通过对Wistar大鼠睁眼前初级视皮层2/3层锥体神经元突触AMPA（α-氨基-3-羧基-5-甲基异恶唑-4-丙酸）介导的微小兴奋性突触后电流 (mEPSCs)的测定分析,研究突触自身稳态可塑性在生后早期初级视皮层的作用特点。方法: 采用红外可视膜片钳技术全细胞模式记录生后4-11(d)（P4-11）Wistar大鼠初级视皮层脑片2/3层锥体神经元AMPA介导的mEPSCs,钳制电位-70 mV。人工脑脊液中加入河豚毒素（TTX）、荷包牡丹碱（BMI）及2-氨基-5-磷酸基戊酸（AP-5）分离出AMPA介导的mEPSCs,加入阻断剂6-氰基-7-硝基喹喔啉-2,3二酮 (CNQX）可消除mEPSCs。使用Clampfit 9.0进行数据分析。结果: P4至P11,大鼠初级视皮层2/3层锥体神经元AMPA介导的mEPSCs的波幅呈现上升趋势,频率自P7至P11逐渐增加,上升时间常数及下降时间常数均呈缩短趋势,以下降时间常数变化为著。P4至P7可见“单通道样”电流形态。结论: 在大鼠睁眼前初级视皮层2/3层锥体神经元亦存在突触自身稳态可塑性调节机制,其作用特点不同于睁眼后。     

运动疲劳损害大鼠空间认知能力并导致海马CA1区L-LTP抑制

目的:探讨运动疲劳对空间认知能力的影响及海马突触可塑性调控机制。方法:采用随机数字法将雄性SD大鼠分为对照组(control)和疲劳组(fatigue),选用3级递增负荷跑台训练方案,建立慢性力竭运动疲劳模型。利用Y迷宫空间识别记忆实验评估大鼠的空间识别和记忆变化,使用Western Blot测定海马组织cAMP反应元件结合蛋白(CREB)表达及磷酸化水平,并利用在体电生理记录大鼠海马CA1区晚期时相长时程增强效应(L-LTP),随后通过免疫组织化学染色观察大鼠海马CA1区小清蛋白(PV)的表达。结果:疲劳组大鼠在新异臂的停留时间比和在各臂的总穿梭次数均明显低于对照组(P<0.01)。高频刺激后30、60、120直至180 min,疲劳组大鼠海马CA1区场兴奋性突触后电位(fEPSP)斜率较对照组大鼠均显著降低(P<0.01)。Western Blot结果表明,疲劳组大鼠海马组织磷酸化CREB(p-CREB)水平明显低于对照组(P<0.05)。免疫组织化学染色显示,疲劳组大鼠海马CA1区PV表达下调(P<0.05)。结论:运动疲劳可导致大鼠空间认知能力受损,其机...     

双眼剥夺猫视皮层细胞的某些反应特性

小猫出生后第四周开始将其双眼剥夺,成年后记录。以正弦光栅为刺激研究了这些猫纹状皮层细胞的单眼和双眼反应特性。结果说明:在发育过程中,出生后早期视觉经验,对于保持相当比例的皮层细胞对视差调谐,是重要的。同样地,正常视觉环境刺激,对于保持双眼匹配的空间频率和朝向调谐是必需的。     

Reactivation of visual cortical plasticity by NEP1-40 from early monocular deprivation in adult rats

Amblyopia is difficult to cure in adult due to the declination of visual cortical plasticity with age. However, the mechanisms limiting adult cortical plasticity are still unclear. Inhibition factors associated with myelin are suggested to be crucial for the ocular dominance plasticity in the visual cortex. We hypothesize that blocking Nogo-NgR system with NEP1–40 in adult visual cortex will reactivate the structural and functional plasticity. To back up this hypothesis, we subjected postnatal day 21 (P21) rats to monocular deprivation (MD) model until P45. Then the deprived eyes of MD model rats were reopened and followed by NEP1–40 or PBS administration for 7 days. Dendritic spine densities, ultrastructral modifications of synaptic junctions and objective visual function were examined at P52 to determine the therapeutic effects of NEP1–40. Our findings suggest a new curative role for NEP1–40 in structural and functional recovery from the deficits of adult MD rats, and offer a potential therapeutic tool for curing amblyopia and other cortically based visual disorders.     

Parvalbumin immunoreactivity: a reliable marker for the effects of monocular deprivation in the rat visual cortex.

In mammals, monocular deprivation performed during the early stages of postnatal development (critical period) dramatically affects the functional organization of the visual cortex. Since the early work of Hubel and Wiesel, the effects of monocular deprivation are accounted for by the fibers driven by the two eyes competing for the control of cortical territories. In cat and monkey striking structural changes accompany the functional effects of monocular deprivation. Also, in the rat, monocular deprivation causes functional alteration at the level of visual cortex; no structural correlates of these effects, however, have so far been described. Parvalbumin is a calcium binding protein that in the neocortex colocalizes with a subpopulation of GABAergic neurons. Here we report that in the rat monocular deprivation results in a dramatic reduction of parvalbumin-like immunoreactivity in the visual cortex contralateral to the deprived eye. This effect is due to competitive phenomena and not to visual deprivation itself, it is restricted to the binocular portion of the visual cortex and neither binocular deprivation, nor dark rearing can induce it. We conclude that parvalbumin-like immunoreactivity is a useful immunohistochemical marker for the effects of monocular deprivation in the rat visual cortex.     

Molecular mechanism for loss of visual cortical responsiveness following brief monocular deprivation

A dramatic form of experience-dependent synaptic plasticity is revealed in visual cortex when one eye is temporarily deprived of vision during early postnatal life. Monocular deprivation (MD) alters synaptic transmission such that cortical neurons cease to respond to stimulation of the deprived eye, but how this occurs is poorly understood. Here we show in rat visual cortex that brief MD sets in motion the same molecular and functional changes as the experimental model of homosynaptic long-term depression (LTD), and that prior synaptic depression by MD occludes subsequent induction of LTD. The mechanisms of LTD, about which there is now a detailed understanding, therefore contribute to visual cortical plasticity.     

Differential regulation of synapsin phosphorylation by monocular deprivation in juveniles and adults

The rodent visual cortex retains significant ocular dominance plasticity beyond the traditional postnatal critical period. However, the intracellular mechanisms that underlie the cortical response to monocular deprivation are predicted to be different in juveniles and adults. Here we show monocular deprivation in adult, but not juvenile rats, induced an increase in the phosphorylation of the prominent presynaptic effecter protein synapsin at two key sites known to regulate synapsin function. Monocular deprivation in adults induced an increase in synapsin phosphorylation at the PKA consensus site (site 1) and the CaMKII consensus site (site 3) in the visual cortex ipsilateral to the deprived eye, which is dominated by non-deprived eye input. The increase in synapsin phosphorylation was observed in total cortical homogenate, but not synaptoneurosomes, suggesting that the pool of synapsin targeted by monocular deprivation in adults does not co-fractionate with excitatory synapses. Phosphorylation of sites 1 and 3 stimulates the release of synaptic vesicles from a reserve pool and increases in the probability of evoked neurotransmitter release, which may contribute to the strengthening of the non-deprived input characteristic of ocular dominance plasticity in adults.     

Expression of the transcription factor Zif268 in the visual cortex of monocularly deprived rats: effects of nerve growth factor.

Neurotrophins are known to be involved in experience-dependent plasticity of the visual cortex. Here, we have characterized in detail the effects of intraventricular nerve growth factor infusion in monocularly deprived rats by using immunostaining for the immediate-early gene product Zif268 as a marker of functional activity with cellular resolution. We have taken advantage of the rapid regulation of Zif268 by visual input to reveal the cortical units that are responsive to the deprived eye after a period of monocular deprivation. We found that responses to the deprived eye were significantly preserved in the cortex of monocularly deprived rats infused with nerve growth factor. The effects of nerve growth factor were greater for cortical cells located in deep layers and with more peripheral receptive fields. Results from Zif268 staining correlated very well with those obtained by single-cell recordings from the visual cortex. Our results demonstrate that exogenous nerve growth factor preserves the functional input from the deprived eye, enabling cortical neurons to activate immediate-early gene expression in response to stimulation of the deprived eye. Furthermore, we show that the intraventricular infusion of nerve growth factor differentially affects the ocular dominance of cells at various depths and eccentricities in the developing cortex.     

Apoptosis and retinal projections in the dorsal lateral geniculate nucleus after monocular deprivation during the later phase of the critical period in the rat

The visual cortex in the rat is matured physiologically by postnatal day 30, but the visual system retains the potential to be reorganized until postnatal day 45. Therefore, we defined the period from postnatal days 28-45 as the ‘late critical phase’. To examine whether monocular deprivation during the late critical phase gives rise to neuronal apoptosis in the dorsal lateral geniculate nucleus (dLGN), we used the terminal deoxyribo-nucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling method and anterograde tracing. The number of apoptotic cells in the dLGN after monocular deprivation at postnatal day 28 showed little difference from control at postnatal day 29, but was significantly increased to more than fourfold of control ipsilaterally to the monocular deprivation at postnatal day 35 and to more than 10-fold of control bilaterally at postnatal day 40. In the control, there were almost no apoptotic neurons in the dLGN on either side at postnatal day 40. In the nucleus ipsilateral to the monocular deprivation, approximately half the apoptotic neurons were found in an area that did not receive a retinal projection. These findings suggest that the biological process of increased apoptosis in the dLGN of rats that received monocular deprivation in the late critical phase may be different from that in the early critical phase. The increased number of apoptotic cells in the dLGN in the late critical phase may not be simply the result of monocular deprivation.     

Effects of monocular deprivation on the expression pattern of alpha-1 and beta-1 adrenergic receptors in the kitten visual cortex

To examine how adrenergic receptors are regulated by experimental manipulation of sensory afferents, we performed immunohistochemical analysis on alpha1-, and beta1-adrenergic receptors in the brain of kittens. In normal development, these receptors were similarly expressed in both hemispheres of the occipital and frontal cortices. Notably, monocular deprivation during the sensitive period of ocular dominance plasticity significantly increased beta1-adrenergic receptor immunoreactivity in the visual cortex ipsilateral to the deprived eye. No increase in the intensity of the immunoreactivity for beta1-adrenergic receptors following monocular deprivation was found in the frontal and parietal regions of the cerebral cortex and subcortical structures, including the lateral geniculate nucleus and superior colliculus. Furthermore, such hemispheric change was not found in the alpha1-adrenergic receptor immunoreactivity following monocular deprivation. Comparisons of images, obtained by double staining for microtubule-associated protein-2 or glial fibrillary acidic protein, indicated that the increased immunoreactivity was localized on both apical dendrites of deep layer neurons and glial cells. These results indicate that the monocular deprivation during the sensitive period of ocular dominance plasticity modified beta1-adrenergic receptor immunoreactivity, including that in glial cells. Therefore, it was suggested that beta1-adrenergic receptors in the glial cells also play important roles in the regulation of ocular dominance plasticity.     

Short‐term monocular deprivation alters early components of visual evoked potentials

Key points

• Short‐term monocular deprivation in adult humans produces a perceptual boost of the deprived eye reflecting homeostatic plasticity.
• Visual evoked potentials (VEPs) to transient stimuli change after 150 min of monocular deprivation in adult humans.
• The amplitude of the C1 component of the VEP at a latency of about 100 ms increases for the deprived eye and decreases for the non‐deprived eye after deprivation, the two effects being highly negatively correlated.
• Similarly, the evoked alpha rhythm increases after deprivation for the deprived eye and decreases for the non‐deprived eye.
• The data demonstrate that primary visual cortex excitability is altered by a short period of monocular deprivation, reflecting homeostatic plasticity.

Abstract

Very little is known about plasticity in the adult visual cortex. In recent years psychophysical studies have shown that short‐term monocular deprivation alters visual perception in adult humans. Specifically, after 150 min of monocular deprivation the deprived eye strongly dominates the dynamics of binocular rivalry, reflecting homeostatic plasticity. Here we investigate the neural mechanisms underlying this form of short‐term visual cortical plasticity by measuring visual evoked potentials (VEPs) on the scalp of adult humans during monocular stimulation before and after 150 min of monocular deprivation. We found that monocular deprivation had opposite effects on the amplitude of the earliest component of the VEP (C1) for the deprived and non‐deprived eye stimulation. C1 amplitude increased (+66%) for the deprived eye, while it decreased (−29%) for the non‐deprived eye. Source localization analysis confirmed that the C1 originates in the primary visual cortex. We further report that following monocular deprivation, the amplitude of the peak of the evoked alpha spectrum increased on average by 23% for the deprived eye and decreased on average by 10% for the non‐deprived eye, indicating a change in cortical excitability. These results indicate that a brief period of monocular deprivation alters interocular balance in the primary visual cortex of adult humans by both boosting the activity of the deprived eye and reducing the activity of the non‐deprived eye. This indicates a high level of residual homeostatic plasticity in the adult human primary visual cortex, probably mediated by a change in cortical excitability.

Abbreviations

EEG

electroencephalography

ERP

event related potential

TMS

transcranial magnetic stimulation

VEP

visual evoked potential