碘缺乏及甲状腺功能减退对大鼠仔鼠海马神经发育的影响及Caveolin-1和Doublecortin等相关蛋白表达机制的研究
|
摘要
目的
碘是人体内一种重要的微量元素,是甲状腺激素(thyroid hormone, TH)合成的重要原料,并且影响甲状腺激素合成与分泌的若干中间环节,甲状腺功能的正常发挥直接受膳食中碘含量的调节。人类生存环境中缺碘可造成碘缺乏病,包括地方性甲状腺肿、克汀病、亚克汀病等。碘缺乏是导致人类智力障碍的重要原因之一。甲状腺激素是正常生长发育所必需的激素,具有促进组织分化、生长与发育成熟的作用,对脑的发育尤为重要。在碘缺乏病区普遍存在孕妇孕期碘供应不足的状况,母体碘的缺乏可直接导致子代脑发育期甲状腺激素供应不足,致使子代生长发育延迟,尤其是中枢神经系统的发育分化障碍。尽管我国已全面实施补碘多年,基本控制了地方性甲状腺肿及克汀病,但在原碘缺乏病区仍有大量碘缺乏导致的亚克汀病患儿存在,碘缺乏对儿童智力和体格广泛及严重的危害性已成为严重影响人口素质,阻碍发展的重大公共卫生问题。在生长发育过程中,海马组织终生存在神经可塑性现象；海马神经发育的改变,伴随着海马相关功能的改变。甲状腺功能减退能损害神经系统的发育和功能,而且碘缺乏能够引起子代中枢神经系统不可逆的损伤,这些机制都不是很明确。Caveolin-1, Synaptophysin, Doublecortin及Neural cell adhesion molecule (NCAM)在生长发育过程中发挥重要的作用：Caveolin-1在细胞迁移中调节细胞膜的成分和细胞表面的扩张,参与信号分子的极化和细胞骨架的重构；Synaptophysin是脑发育过程中常用的反应突触状态的指标,它一直被作为轴突终末特异性标志物,用以检测突触的密度和分布；Doublecortin通过调节新生神经元微管的细胞骨架结构和稳定性,促进未成熟神经元的迁移和分化；NCAM通过亲同性及亲异性结合,调节细胞轴突生长、迁移及目标识别。
碘缺乏和甲状腺功能减退影响大鼠仔鼠海马发育过程中是否涉及到Caveolin-1, Synaptophysin, Doublecortin及NCAM-180的改变,CA1区、CA3区和DG区这几种蛋白的变化规律如何,各区的变化是否不同,目前尚不清楚。据此,我们采用碘缺乏饲料和不同剂量的丙基硫尿嘧啶(Propylthiouracil, PTU)饮水建立碘缺乏和甲状腺功能减退动物模型,观察低甲状腺素状态下海马CA1区、CA3区和DG区的受损状况,应用Western Blotting技术检测不同发育阶段仔鼠海马CA1区、CA3区和DG区Caveolin-1, Synaptophysin, Doublecortin及NCAM-180的变化规律,探讨碘缺乏和甲状腺功能减退导致神经系统发育损害的机制,为碘缺乏性智力低下性疾病的预防提供实验基础和理论依据。
实验方法
1、实验动物与分组
健康2月龄雌性Wistar大鼠,体重250 g-280 g。取孕鼠28只,按体重均衡原则随机分为4组,每组7只孕鼠,分组及饮食为：碘缺乏组,缺碘饲料+自来水；5 ppm PTU组,普通饲料+5 ppm PTU+自来水；15ppm PTU组,普通饲料+15 ppm PTU+自来水；对照组,普通饲料+自来水。自妊娠第6天(Gestational day 6, GD6)起,按照上述方式喂养,直至仔鼠出生后第28天(Postnatal day 28, PN28);此后至PN42,各组均饲以普通饲料,饮用自来水。PN4时,每窝按雌雄数量尽可能相等的原则保留9-10只仔鼠。PN25时断乳、分笼饲养。PN14、PN21、PN28和PN42时,每组随机从不同窝中各抽取8只仔鼠,其中5只仔鼠灌流固定大脑,用于大脑组织病理切片和形态学观察；另3只仔鼠剥离大脑组织,用于Western Blotting测定。
2、仔鼠体重及血清TSH、TH测定
分别于PN14、PN21、PN28和PN42时称取仔鼠体重。在PN14、PN21、PN28和PN42时,每组分别随机选取8只仔鼠,开胸采血,3000 rpm离心5 min,取上清。采用固相化学免疫发光法测定血清中游离四碘甲状腺原氨酸(FT4)、游离三碘甲状腺原氨酸(FT3)和促甲状腺激素(TSH)的含量。
3、海马CA1区、CA3区和DG区存活神经元观察
各组于PN14、PN21、PN28和PN42分别取5只大鼠,采血后,固定大脑,常规石蜡包埋,行冠状位连续切片(6μm),当观察到切片包含完整的海马CA1区、CA3区和DG区时,每隔5～6张连续切片收集1张,制成石蜡切片。每只仔鼠随机选取3张切片,行尼氏染色(Nissl Staining),镜下观察CA1区、CA3区和DG区神经元形态结构,计数每个视野下存活神经元数,取其平均值。
4、海马CA1区、CA3区和DG区神经纤维观察
各组于PN14、PN21、PN28和PN42分别取5只大鼠,采血后,固定大脑,常规石蜡包埋,行冠状位连续切片(6μm),当观察到切片包含完整的海马CA1区、CA3区和DG区时,每隔5～6张连续切片收集1张,制成石蜡切片。每只仔鼠随机选取3张切片,行镀银染色(Silver Staining),镜下观察CA1区、CA3区和DG区神经纤维形态结构及其变化规律。
5、海马CA1区、CA3区和DG区相关蛋白检测分析
在PN14、PN21、PN28和PN42时,每组分别从不同窝中随机选取3只仔鼠,取大脑组织,剥离海马,按其组织学结构切分为CA1区、CA3区和DG区。采用Western Blotting技术分别检测各区Caveolin-1, Synaptophysin, Doublecortin及NCAM-180的表达。
6、统计学处理
采用SPSS12.0建立数据库,进行统计分析。结果表示为Mean±SD。Caveolin-1, Synaptophysin, Doublecortin及NCAM-180表达的主效应分析采用3×4×4析因设计方差分析；在PN14、PN21、PN28和PN42时,各组仔鼠体重,CA1区、CA3区和DG区存活细胞数,Caveolin-1, Synaptophysin, Doublecortin及NCAM-180表达的比较均采用单因素方差分析(ANOVA)。满足方差齐性时,两两比较采用最小显著差值法(Least-significant Difference, LSD);不满足方差齐性时,两两比较采用Dunnett's T3法。P<0.05时差异有统计学意义,方差齐性检验水准定为0.1。
结果
1、大鼠仔鼠激素含量的变化
在PN14-PN28期间,碘缺乏组、5 ppm PTU处理组和15 ppm PTU处理组仔鼠的甲状腺激素含量较对照组明显下降,差别有统计学意义(P<0.05),而TSH含量则反馈性较对照组升高；提示甲状腺功能减退。停止碘缺乏饲料及PTU饮水后,PN42时观察到激素水平基本恢复(P>0.05)。
2、大鼠仔鼠体重的变化
对照组子代大鼠体重增长较快,碘缺乏组及PTU处理组(5 ppm和15 ppm)子代大鼠体重增长慢。对照组仔鼠的体重均显著高于碘缺乏组、5 ppm PTU处理组和15 ppm PTU处理组,差别有统计学意义(P<0.05)。
3、碘缺乏与甲状腺功能减退对仔鼠海马神经元的影响
在PN14时,对照组仔鼠海马CA1区、CA3区和DG区神经元形态正常,胞体排列整齐、紧密,胞膜和核仁清晰,尼氏小体密集,神经纤维数量少；碘缺乏组、5 ppm PTU处理组和15 ppm PTU处理组CA1区、CA3区和DG区神经元形态无明显病理损伤,较对照组无明显差异；神经元计数显示各组间存活细胞数差别无统计学意义(P>0.05)。
在PN21,PN28和PN42时,与对照组相比,镜下可见碘缺乏组、5 ppm PTU处理组和15 ppm PTU处理组CA1区、CA3区和DG区神经元形态较规则,胞体排列稀疏、较整齐,神经纤维数量少；其中PN42时,可见神经纤维排列紊乱、扭曲或断裂现象；神经元计数显示实验组较对照组存活细胞数少,差别有统计学意义(P<0.05)。
4、碘缺乏与甲状腺功能减退对仔鼠海马Caveolin-1Synaptophysin、Doublecortin及NCAM-180表达的影响
在PN14,PN21,PN28和PN42时,仔鼠海马CA1区、CA3区和DG区的Western Blotting结果显示,Caveolin-1表达碘缺乏组和15 ppm PTU处理组较对照组明显增多,差别有统计学意义(P<0.05),5 ppm PTU处理组虽较对照组增多,但仅CA1区和CA3区部分时间点差别有统计学意义；Synaptophysin表达碘缺乏组和15 ppm PTU处理组较对照组明显降低,差别有统计学意义(P<0.05),5ppmPTU处理组与对照组相比,差别无统计学意义(P>0.05); Doublecortin表达碘缺乏组和15 ppm PTU处理组较对照组明显降低,差别有统计学意义(P<0.05),5 ppm PTU处理组虽较对照组降低,但仅部分时间点差别有统计学意义；NCAM-180表达碘缺乏组和15 ppm PTU处理组较对照组明显增多,差别有统计学意义(P<0.05),5 ppm PTU处理组虽较对照组增多,但仅DG区个别时间点差别有统计学意义。
结论
1、用碘缺乏饲料和不同剂量PTU饮水建立甲状腺功能减退大鼠仔鼠动物模型,发现仔鼠海马CA1区、CA3区和DG区存活神经细胞数减少,神经纤维受损。
2、碘缺乏和甲状腺功能减退可导致Caveolin-1和NCAM-180表达增多,Doublecortin和Synaptophysin表达下降。
3、即使PN42时TH和TSH水平基本恢复,但神经元损伤及蛋白水平的异常改变仍然存在。推测碘缺乏和甲状腺功能减退损伤仔鼠海马神经发育的机制可能是由于其上调海马区Caveolin-1和NCAM-180的蛋白水平及下调Doublecortin和Synaptophysin的蛋白水平。
Iodine is an essential trace element that is critical for the synthesis of thyroid hormone (TH). The maintenance of thyroid function directly depends on adequate availability of dietary iodine. Iodine deficiency is one of the most common, preventable causes of brain damage in the world. Sufficient levels of TH, thyroxine (T4), and 3,5,3-triiodothyronine (T3) are indispensable to mammalian brain development and metabolic homeostasis, and they play a vital role in hippocampal formation; the hippocampus expresses receptors for TH. It is clear that hypothyroidism results in stunted growth and impairs brain development. Caveolin-1 plays an important role in the formation of plasma membrane caveolae and anchors them to the actin cytoskeleton, regulates cell interactions with the extracellular matrix, pulls together and modulates signaling molecules, and transports cholesterol. Moreover, caveolin-1 may participate in neuronal differentiation and maturation because it is localized to the growth cones and dendrites of hippocampal neurons. Synaptophysin is an integral protein of small pre-synaptic vesicles that is involved in the release of neurotransmitter vesicles and present in virtually all nerve terminals.The levels of synaptophysin protein expression parallel the numbers of synapses and the density and quantity of nerve terminals. Doublecortin could regulate the migration of multiple classes of cortical neurons, facilitating migration via actions at the distal ends of neurites that promote neurite extension. Neural cell adhesion molecule (NCAM)-180 has been implicated in critical morphogenetic processes during CNS development such as neuronal migration and layering, and axonal growth, guidance and fasciculation. Importantly, researches have determined that iodine deficiency-induced developmental defects of the CNS are irreversible in fetuses and children, and hypothyroidism alteres synaptic development and function. However, the underlying mechanisms are not known. Thus, caveolin-1, synaptophysin, doublecortin and NCAM-180 may be involved in the deficits in neural development that follow iodine deficiency and hypothyroidism. The present study provides an important complement to the existing literature regarding alterations in hippocampal development induced by iodine deficiency and PTU-induced hypothyroidism.
Methods
1、Animals
Wistar rats (250-280 g) were obtained from the Center for Experimental Animals at China Medical University (Shenyang, China). Rats were housed at an environmental temperature of 24±1℃on a 12/12-h light/dark cycle and had access to food and water ad libitum. The day the vaginal plug was discovered was considered to be gestational day (GD) 0. The pregnant rats were randomly assigned into four treatment groups (n= 7 per dose group):control group, iodine-deficient group,5 ppm PTU-treated group, or 15 ppm PTU-treated group. The iodine-deficient group was administered with an iodine-deficient diet (iodine content:14.11±1.96 ng/g) and tap water from GD6 until postnatal day (PN) 28. Rats of the PTU-treated groups were administered 5 ppm or 15 ppm PTU in the drinking water and fed with a normal diet (iodine content: 470.50±46.52 ng/g) from GD6 to PN28. Each litter was culled to 9-10 pups on PN4 (equal numbers of males and females in each group when possible). On PN14, PN21, PN28, or PN42,8 pups in each group were weighted and anesthetized by etherization.
2、Observation of body weight and measurement of FT3, FT4, and TSH
During growth and development, all the pups were observed carefully on the body weight. Heart blood samples were obtained from 8 pups in each group at each time point, and serum was separated and stored at-70℃for subsequent measurement of free triiodothyronine (FT3), free thyroxine (FT4), and thyroid stimulating hormone (TSH) by a supersensitive chemiluminescence immunoassay.
3、Measurement of surviving neurons
On PN14, PN21, PN28, and PN42, the brains of 5 rats per group were preserved via intracardiac perfusion with 50-100 ml normal saline containing 0.02% heparin, followed by 200-400ml 4% paraformaldehyde in 0.1 M potassium phosphate buffer (pH 7.4). Then, the brains were quickly removed from the skull and fixed overnight in the same fixative. The fixed brains were embedded in paraffin and sectioned into 6-μm-thick coronal sections. Brains were sectioned in a serial manner when intact structure of hippocampus was observed in the slices. Every fifth/sixth slice was collected per animal on gelatin-coated microscope slides. Three sections of each rat brain were selected randomly for Nissl staining. After deparaffinization in xylene for 10 min followed by 100% ethanol, slides were washed in deionized water. Then, slides were performed with routine Nissl staining based on the thionine technique and then analyzed under a microscope. The hippocampal subregions of interest were selected: CA1, CA3, and dentate gyrus (DG). Imaging-Pro-Plus (Media Cybernetics, Inc., Silver Spring, MD) was used to perform quantitative analysis of cell number. Total number of surviving cells was counted in a blinded manner only if structures were identified clearly. Three different fields were selected from CA1, CA3, or DG regions per section respectively and three sections per animal were evaluated to obtain a mean value. Five rats per group per time point were used to obtain an overall mean value for subsequent statistical analysis.
4、Analysis of nerve fibers
On PN14, PN21, PN28, and PN42, the brains of 5 rats per group were preserved via intracardiac perfusion with 50-100 ml normal saline containing 0.02% heparin, followed by 200-400ml 4% paraformaldehyde in 0.1 M potassium phosphate buffer (pH 7.4). Then, the brains were quickly removed from the skull and fixed overnight in the same fixative. The fixed brains were embedded in paraffin and sectioned into 6-μm-thick coronal sections. Brains were sectioned in a serial manner when intact structure of hippocampus was observed in the slices. Every fifth/sixth slice was collected per animal on gelatin-coated microscope slides. Three sections of each rat brain were selected, and then they were stained by silver staining. The sections were microscopically analyzed at 40×magnification for the hippocampal sub-regions of interest:CA1, CA3, and DG. The boundaries for the sub-regions were determined according to the Paxions-Wastson atlas of the rat brain. Then images were obtained at a magnification of 400×to analyze the nerve fibers and cell bodies.
5、Detection of related proteins
The brains of the 3 pups per group per time point were rapidly dissected from the skull and immediately submerged in ice-cold artificial cerebrospinal fluid. Then, the hippocampus was removed and dissected on ice rapidly. With the help of a dissecting microscope, the hippocampus was bisected and the dorsomedial half was divided into four slabs cut perpendicular to the long axis of the hippocampus. According to the Paxions-Wastson atlas of the rat brain, each slab was dissected into CA1, CA3 and DG regions. The protein was estimated by coomassie brilliant blue staining. Proteins were transferred onto nitrocellulose membranes. After blocking the nonspecific sites with PBS containing 0.1% Tween 20 and 5% defatted dried milk, membranes were washed and incubated with primary antibody, rabbit anti-caveolin-1, rabbit anti-synaptophysin, rabbit anti-doublecortin, rabbit anti-NCAM and rabbit anti-β-actin. Blots were developed with the Easy Enhanced Chemiluminescence Western Blot Kit. For each blot, the P-actin lanes were analyzed as a quality control sample. The signals from target bands on each blot were normalized to the mean signal for the quality control sample bands in order to simplify the comparisons across the blots and reduce inter-blot variability.
6、Statistical analysis
All analyses were carried out using SPSS software, and the analyzers were blind to the treatment of each group. Data are presented as mean±standard deviation (SD) and considered statistically significant at P＜0.05. To verify consistent protein loading among the gels, some blots were probed for P-actin and then the ratio with caveolin-1 or synaptophysin or doublecortin or NCAM was determined. There was no statistical difference between the density percent control and the ratio with P-actin; therefore, the analyses for the percent control are presented for each blot. At each time point, comparisons of the pups'body weights, FT3, FT4 and TSH levels, surviving cell numbers, protein levels of caveolin-1, synaptophysin, doublecortin and NCAM-180 were made using one-way ANOVA. When the F-value indicated significance, least-significant difference post hoc comparisons were made as appropriate to correct for multiple comparisons. All P-values were two-tailed.
Results
1、Changes of TH and TSH
Reductions in TH with concomitant elevations in TSH were observed in hypothyroid offspring by the researcher. Interestingly and in line with this statement, our data showed that the offspring displayed hypothyroxinemia in iodine-deficient group (reduced FT4 with no significant increase in TSH) and hypothyroidism in 5ppm and 15ppm PTU-treated groups. The iodine-deficient and 15ppm PTU-treated groups had significantly lower serum FT3 and FT4 than the controls on PN14, PN21, and PN28 (P＜0.05). TSH levels were increased significantly in 5ppm and 15ppm PTU-treated offspring than controls (P＜0.05). On PN42, the concentrations of serum FT3, FT4, and TSH in iodine-deficient and PTU-treated groups were restored.
2、Alterations of body weights
Many studies have shown that PTU treatment reduces offspring body weights. Our data shown that the offspring's body weights in iodine-deficient,5ppm and 15ppm PTU-treated groups were statistically significant lower than those of controls on PN14, PN21, PN28, and PN42 (P＜0.05)
3、Impairment of neurons
Iodine deficiency and hypothyroidism reduce hippocampal surviving cells. To investigate whether or not hippocampal neuronal survival was impaired by iodine deficiency and hypothyroidism, histological examination of hippocampal neurons was performed on Nissl-stained sections. The results reveal lower surviving cells in the hippocampal neurons of offspring with low circulating TH levels in the CA1, CA3, and DG regions on PN21, PN28, and PN42(P＜0.05). The mean number of surviving cells in the hippocampus of the iodine-deficient,5 ppm PTU-treated, and 15 ppm PTU-treated rats was reduced compared to controls. On PN28 and PN42, compared with the control group, nerve fibers were found injured in the treated groups of regions CA3 and DG, as well as reduced in number. Injury appeared as fibers broken, disordered, bundled together, or fused with other fibers. In the control group, healthy nerve fibers were observed in all regions. The control nerve fibers were arranged in a good order with clear processes. Unlike regions CA3 and DG, nerve fibers in region CA1 were not yet damaged on PN28.
4、Effects on the related proteins
On PN14, PN21, PN28, and PN42, caveolin-1, synaptophysin, doublecortin and NCAM-180 were expressed in the CA1, CA3, and DG regions of the hippocampus of all groups, respectively. Significant upregulation of caveolin-1 was observed in rats exposed to the iodine-deficient,5 ppm PTU-adulterated, or 15 ppm PTU-adulterated diet relative to controls (P＜0.05). Synaptophysin was significantly down-regulated in rats exposed to the iodine-deficient,5 ppm PTU-adulterated, or 15 ppm PTU-adulterated diet relative to the controls (P＜0.05). A significant downregulation of doublecortin was observed in rats exposed to iodine-deficient and 15 ppm PTU-treated groups in CA1, CA3, and DG regions (P＜0.05). Upregulation of NCAM-180 in CA1, CA3, and DG regions were observed in iodine-deficient and 15 ppm PTU-treated rats from PN14 on (P＜0.05). Especially on PN42, we observed doublecortin decrease and NCAM-180 increase when TH levels were restored to a normal state, suggesting irreversible hippocampus impairment.
Conclusion
1、Iodine deficiency or PTU-induced hypothyroidism led to morphological damage in the hippocampus. Lower surviving cells in the hippocampal neurons of offspring with low circulating TH levels in the hippocampal subregions and nerve fibers were impaired.
2、Developmental iodine deficiency or PTU-induced hypothyroidism led to downregulation of doublecortin and synaptophysin and upregulation of Caveolin-1 and NCAM-180 in the hippocampal subregions at each time point.
3、The alterations of hippocampal morphology and expression of related proteins were still obvious on PN42 despite the recovery of TH to normal status.
碘是人体内一种重要的微量元素,是甲状腺激素(thyroid hormone, TH)合成的重要原料,并且影响甲状腺激素合成与分泌的若干中间环节,甲状腺功能的正常发挥直接受膳食中碘含量的调节。人类生存环境中缺碘可造成碘缺乏病,包括地方性甲状腺肿、克汀病、亚克汀病等。碘缺乏是导致人类智力障碍的重要原因之一。甲状腺激素是正常生长发育所必需的激素,具有促进组织分化、生长与发育成熟的作用,对脑的发育尤为重要。在碘缺乏病区普遍存在孕妇孕期碘供应不足的状况,母体碘的缺乏可直接导致子代脑发育期甲状腺激素供应不足,致使子代生长发育延迟,尤其是中枢神经系统的发育分化障碍。尽管我国已全面实施补碘多年,基本控制了地方性甲状腺肿及克汀病,但在原碘缺乏病区仍有大量碘缺乏导致的亚克汀病患儿存在,碘缺乏对儿童智力和体格广泛及严重的危害性已成为严重影响人口素质,阻碍发展的重大公共卫生问题。在生长发育过程中,海马组织终生存在神经可塑性现象；海马神经发育的改变,伴随着海马相关功能的改变。甲状腺功能减退能损害神经系统的发育和功能,而且碘缺乏能够引起子代中枢神经系统不可逆的损伤,这些机制都不是很明确。Caveolin-1, Synaptophysin, Doublecortin及Neural cell adhesion molecule (NCAM)在生长发育过程中发挥重要的作用：Caveolin-1在细胞迁移中调节细胞膜的成分和细胞表面的扩张,参与信号分子的极化和细胞骨架的重构；Synaptophysin是脑发育过程中常用的反应突触状态的指标,它一直被作为轴突终末特异性标志物,用以检测突触的密度和分布；Doublecortin通过调节新生神经元微管的细胞骨架结构和稳定性,促进未成熟神经元的迁移和分化；NCAM通过亲同性及亲异性结合,调节细胞轴突生长、迁移及目标识别。
碘缺乏和甲状腺功能减退影响大鼠仔鼠海马发育过程中是否涉及到Caveolin-1, Synaptophysin, Doublecortin及NCAM-180的改变,CA1区、CA3区和DG区这几种蛋白的变化规律如何,各区的变化是否不同,目前尚不清楚。据此,我们采用碘缺乏饲料和不同剂量的丙基硫尿嘧啶(Propylthiouracil, PTU)饮水建立碘缺乏和甲状腺功能减退动物模型,观察低甲状腺素状态下海马CA1区、CA3区和DG区的受损状况,应用Western Blotting技术检测不同发育阶段仔鼠海马CA1区、CA3区和DG区Caveolin-1, Synaptophysin, Doublecortin及NCAM-180的变化规律,探讨碘缺乏和甲状腺功能减退导致神经系统发育损害的机制,为碘缺乏性智力低下性疾病的预防提供实验基础和理论依据。
实验方法
1、实验动物与分组
健康2月龄雌性Wistar大鼠,体重250 g-280 g。取孕鼠28只,按体重均衡原则随机分为4组,每组7只孕鼠,分组及饮食为：碘缺乏组,缺碘饲料+自来水；5 ppm PTU组,普通饲料+5 ppm PTU+自来水；15ppm PTU组,普通饲料+15 ppm PTU+自来水；对照组,普通饲料+自来水。自妊娠第6天(Gestational day 6, GD6)起,按照上述方式喂养,直至仔鼠出生后第28天(Postnatal day 28, PN28);此后至PN42,各组均饲以普通饲料,饮用自来水。PN4时,每窝按雌雄数量尽可能相等的原则保留9-10只仔鼠。PN25时断乳、分笼饲养。PN14、PN21、PN28和PN42时,每组随机从不同窝中各抽取8只仔鼠,其中5只仔鼠灌流固定大脑,用于大脑组织病理切片和形态学观察；另3只仔鼠剥离大脑组织,用于Western Blotting测定。
2、仔鼠体重及血清TSH、TH测定
分别于PN14、PN21、PN28和PN42时称取仔鼠体重。在PN14、PN21、PN28和PN42时,每组分别随机选取8只仔鼠,开胸采血,3000 rpm离心5 min,取上清。采用固相化学免疫发光法测定血清中游离四碘甲状腺原氨酸(FT4)、游离三碘甲状腺原氨酸(FT3)和促甲状腺激素(TSH)的含量。
3、海马CA1区、CA3区和DG区存活神经元观察
各组于PN14、PN21、PN28和PN42分别取5只大鼠,采血后,固定大脑,常规石蜡包埋,行冠状位连续切片(6μm),当观察到切片包含完整的海马CA1区、CA3区和DG区时,每隔5～6张连续切片收集1张,制成石蜡切片。每只仔鼠随机选取3张切片,行尼氏染色(Nissl Staining),镜下观察CA1区、CA3区和DG区神经元形态结构,计数每个视野下存活神经元数,取其平均值。
4、海马CA1区、CA3区和DG区神经纤维观察
各组于PN14、PN21、PN28和PN42分别取5只大鼠,采血后,固定大脑,常规石蜡包埋,行冠状位连续切片(6μm),当观察到切片包含完整的海马CA1区、CA3区和DG区时,每隔5～6张连续切片收集1张,制成石蜡切片。每只仔鼠随机选取3张切片,行镀银染色(Silver Staining),镜下观察CA1区、CA3区和DG区神经纤维形态结构及其变化规律。
5、海马CA1区、CA3区和DG区相关蛋白检测分析
在PN14、PN21、PN28和PN42时,每组分别从不同窝中随机选取3只仔鼠,取大脑组织,剥离海马,按其组织学结构切分为CA1区、CA3区和DG区。采用Western Blotting技术分别检测各区Caveolin-1, Synaptophysin, Doublecortin及NCAM-180的表达。
6、统计学处理
采用SPSS12.0建立数据库,进行统计分析。结果表示为Mean±SD。Caveolin-1, Synaptophysin, Doublecortin及NCAM-180表达的主效应分析采用3×4×4析因设计方差分析；在PN14、PN21、PN28和PN42时,各组仔鼠体重,CA1区、CA3区和DG区存活细胞数,Caveolin-1, Synaptophysin, Doublecortin及NCAM-180表达的比较均采用单因素方差分析(ANOVA)。满足方差齐性时,两两比较采用最小显著差值法(Least-significant Difference, LSD);不满足方差齐性时,两两比较采用Dunnett's T3法。P<0.05时差异有统计学意义,方差齐性检验水准定为0.1。
结果
1、大鼠仔鼠激素含量的变化
在PN14-PN28期间,碘缺乏组、5 ppm PTU处理组和15 ppm PTU处理组仔鼠的甲状腺激素含量较对照组明显下降,差别有统计学意义(P<0.05),而TSH含量则反馈性较对照组升高；提示甲状腺功能减退。停止碘缺乏饲料及PTU饮水后,PN42时观察到激素水平基本恢复(P>0.05)。
2、大鼠仔鼠体重的变化
对照组子代大鼠体重增长较快,碘缺乏组及PTU处理组(5 ppm和15 ppm)子代大鼠体重增长慢。对照组仔鼠的体重均显著高于碘缺乏组、5 ppm PTU处理组和15 ppm PTU处理组,差别有统计学意义(P<0.05)。
3、碘缺乏与甲状腺功能减退对仔鼠海马神经元的影响
在PN14时,对照组仔鼠海马CA1区、CA3区和DG区神经元形态正常,胞体排列整齐、紧密,胞膜和核仁清晰,尼氏小体密集,神经纤维数量少；碘缺乏组、5 ppm PTU处理组和15 ppm PTU处理组CA1区、CA3区和DG区神经元形态无明显病理损伤,较对照组无明显差异；神经元计数显示各组间存活细胞数差别无统计学意义(P>0.05)。
在PN21,PN28和PN42时,与对照组相比,镜下可见碘缺乏组、5 ppm PTU处理组和15 ppm PTU处理组CA1区、CA3区和DG区神经元形态较规则,胞体排列稀疏、较整齐,神经纤维数量少；其中PN42时,可见神经纤维排列紊乱、扭曲或断裂现象；神经元计数显示实验组较对照组存活细胞数少,差别有统计学意义(P<0.05)。
4、碘缺乏与甲状腺功能减退对仔鼠海马Caveolin-1Synaptophysin、Doublecortin及NCAM-180表达的影响
在PN14,PN21,PN28和PN42时,仔鼠海马CA1区、CA3区和DG区的Western Blotting结果显示,Caveolin-1表达碘缺乏组和15 ppm PTU处理组较对照组明显增多,差别有统计学意义(P<0.05),5 ppm PTU处理组虽较对照组增多,但仅CA1区和CA3区部分时间点差别有统计学意义；Synaptophysin表达碘缺乏组和15 ppm PTU处理组较对照组明显降低,差别有统计学意义(P<0.05),5ppmPTU处理组与对照组相比,差别无统计学意义(P>0.05); Doublecortin表达碘缺乏组和15 ppm PTU处理组较对照组明显降低,差别有统计学意义(P<0.05),5 ppm PTU处理组虽较对照组降低,但仅部分时间点差别有统计学意义；NCAM-180表达碘缺乏组和15 ppm PTU处理组较对照组明显增多,差别有统计学意义(P<0.05),5 ppm PTU处理组虽较对照组增多,但仅DG区个别时间点差别有统计学意义。
结论
1、用碘缺乏饲料和不同剂量PTU饮水建立甲状腺功能减退大鼠仔鼠动物模型,发现仔鼠海马CA1区、CA3区和DG区存活神经细胞数减少,神经纤维受损。
2、碘缺乏和甲状腺功能减退可导致Caveolin-1和NCAM-180表达增多,Doublecortin和Synaptophysin表达下降。
3、即使PN42时TH和TSH水平基本恢复,但神经元损伤及蛋白水平的异常改变仍然存在。推测碘缺乏和甲状腺功能减退损伤仔鼠海马神经发育的机制可能是由于其上调海马区Caveolin-1和NCAM-180的蛋白水平及下调Doublecortin和Synaptophysin的蛋白水平。
Iodine is an essential trace element that is critical for the synthesis of thyroid hormone (TH). The maintenance of thyroid function directly depends on adequate availability of dietary iodine. Iodine deficiency is one of the most common, preventable causes of brain damage in the world. Sufficient levels of TH, thyroxine (T4), and 3,5,3-triiodothyronine (T3) are indispensable to mammalian brain development and metabolic homeostasis, and they play a vital role in hippocampal formation; the hippocampus expresses receptors for TH. It is clear that hypothyroidism results in stunted growth and impairs brain development. Caveolin-1 plays an important role in the formation of plasma membrane caveolae and anchors them to the actin cytoskeleton, regulates cell interactions with the extracellular matrix, pulls together and modulates signaling molecules, and transports cholesterol. Moreover, caveolin-1 may participate in neuronal differentiation and maturation because it is localized to the growth cones and dendrites of hippocampal neurons. Synaptophysin is an integral protein of small pre-synaptic vesicles that is involved in the release of neurotransmitter vesicles and present in virtually all nerve terminals.The levels of synaptophysin protein expression parallel the numbers of synapses and the density and quantity of nerve terminals. Doublecortin could regulate the migration of multiple classes of cortical neurons, facilitating migration via actions at the distal ends of neurites that promote neurite extension. Neural cell adhesion molecule (NCAM)-180 has been implicated in critical morphogenetic processes during CNS development such as neuronal migration and layering, and axonal growth, guidance and fasciculation. Importantly, researches have determined that iodine deficiency-induced developmental defects of the CNS are irreversible in fetuses and children, and hypothyroidism alteres synaptic development and function. However, the underlying mechanisms are not known. Thus, caveolin-1, synaptophysin, doublecortin and NCAM-180 may be involved in the deficits in neural development that follow iodine deficiency and hypothyroidism. The present study provides an important complement to the existing literature regarding alterations in hippocampal development induced by iodine deficiency and PTU-induced hypothyroidism.
Methods
1、Animals
Wistar rats (250-280 g) were obtained from the Center for Experimental Animals at China Medical University (Shenyang, China). Rats were housed at an environmental temperature of 24±1℃on a 12/12-h light/dark cycle and had access to food and water ad libitum. The day the vaginal plug was discovered was considered to be gestational day (GD) 0. The pregnant rats were randomly assigned into four treatment groups (n= 7 per dose group):control group, iodine-deficient group,5 ppm PTU-treated group, or 15 ppm PTU-treated group. The iodine-deficient group was administered with an iodine-deficient diet (iodine content:14.11±1.96 ng/g) and tap water from GD6 until postnatal day (PN) 28. Rats of the PTU-treated groups were administered 5 ppm or 15 ppm PTU in the drinking water and fed with a normal diet (iodine content: 470.50±46.52 ng/g) from GD6 to PN28. Each litter was culled to 9-10 pups on PN4 (equal numbers of males and females in each group when possible). On PN14, PN21, PN28, or PN42,8 pups in each group were weighted and anesthetized by etherization.
2、Observation of body weight and measurement of FT3, FT4, and TSH
During growth and development, all the pups were observed carefully on the body weight. Heart blood samples were obtained from 8 pups in each group at each time point, and serum was separated and stored at-70℃for subsequent measurement of free triiodothyronine (FT3), free thyroxine (FT4), and thyroid stimulating hormone (TSH) by a supersensitive chemiluminescence immunoassay.
3、Measurement of surviving neurons
On PN14, PN21, PN28, and PN42, the brains of 5 rats per group were preserved via intracardiac perfusion with 50-100 ml normal saline containing 0.02% heparin, followed by 200-400ml 4% paraformaldehyde in 0.1 M potassium phosphate buffer (pH 7.4). Then, the brains were quickly removed from the skull and fixed overnight in the same fixative. The fixed brains were embedded in paraffin and sectioned into 6-μm-thick coronal sections. Brains were sectioned in a serial manner when intact structure of hippocampus was observed in the slices. Every fifth/sixth slice was collected per animal on gelatin-coated microscope slides. Three sections of each rat brain were selected randomly for Nissl staining. After deparaffinization in xylene for 10 min followed by 100% ethanol, slides were washed in deionized water. Then, slides were performed with routine Nissl staining based on the thionine technique and then analyzed under a microscope. The hippocampal subregions of interest were selected: CA1, CA3, and dentate gyrus (DG). Imaging-Pro-Plus (Media Cybernetics, Inc., Silver Spring, MD) was used to perform quantitative analysis of cell number. Total number of surviving cells was counted in a blinded manner only if structures were identified clearly. Three different fields were selected from CA1, CA3, or DG regions per section respectively and three sections per animal were evaluated to obtain a mean value. Five rats per group per time point were used to obtain an overall mean value for subsequent statistical analysis.
4、Analysis of nerve fibers
On PN14, PN21, PN28, and PN42, the brains of 5 rats per group were preserved via intracardiac perfusion with 50-100 ml normal saline containing 0.02% heparin, followed by 200-400ml 4% paraformaldehyde in 0.1 M potassium phosphate buffer (pH 7.4). Then, the brains were quickly removed from the skull and fixed overnight in the same fixative. The fixed brains were embedded in paraffin and sectioned into 6-μm-thick coronal sections. Brains were sectioned in a serial manner when intact structure of hippocampus was observed in the slices. Every fifth/sixth slice was collected per animal on gelatin-coated microscope slides. Three sections of each rat brain were selected, and then they were stained by silver staining. The sections were microscopically analyzed at 40×magnification for the hippocampal sub-regions of interest:CA1, CA3, and DG. The boundaries for the sub-regions were determined according to the Paxions-Wastson atlas of the rat brain. Then images were obtained at a magnification of 400×to analyze the nerve fibers and cell bodies.
5、Detection of related proteins
The brains of the 3 pups per group per time point were rapidly dissected from the skull and immediately submerged in ice-cold artificial cerebrospinal fluid. Then, the hippocampus was removed and dissected on ice rapidly. With the help of a dissecting microscope, the hippocampus was bisected and the dorsomedial half was divided into four slabs cut perpendicular to the long axis of the hippocampus. According to the Paxions-Wastson atlas of the rat brain, each slab was dissected into CA1, CA3 and DG regions. The protein was estimated by coomassie brilliant blue staining. Proteins were transferred onto nitrocellulose membranes. After blocking the nonspecific sites with PBS containing 0.1% Tween 20 and 5% defatted dried milk, membranes were washed and incubated with primary antibody, rabbit anti-caveolin-1, rabbit anti-synaptophysin, rabbit anti-doublecortin, rabbit anti-NCAM and rabbit anti-β-actin. Blots were developed with the Easy Enhanced Chemiluminescence Western Blot Kit. For each blot, the P-actin lanes were analyzed as a quality control sample. The signals from target bands on each blot were normalized to the mean signal for the quality control sample bands in order to simplify the comparisons across the blots and reduce inter-blot variability.
6、Statistical analysis
All analyses were carried out using SPSS software, and the analyzers were blind to the treatment of each group. Data are presented as mean±standard deviation (SD) and considered statistically significant at P＜0.05. To verify consistent protein loading among the gels, some blots were probed for P-actin and then the ratio with caveolin-1 or synaptophysin or doublecortin or NCAM was determined. There was no statistical difference between the density percent control and the ratio with P-actin; therefore, the analyses for the percent control are presented for each blot. At each time point, comparisons of the pups'body weights, FT3, FT4 and TSH levels, surviving cell numbers, protein levels of caveolin-1, synaptophysin, doublecortin and NCAM-180 were made using one-way ANOVA. When the F-value indicated significance, least-significant difference post hoc comparisons were made as appropriate to correct for multiple comparisons. All P-values were two-tailed.
Results
1、Changes of TH and TSH
Reductions in TH with concomitant elevations in TSH were observed in hypothyroid offspring by the researcher. Interestingly and in line with this statement, our data showed that the offspring displayed hypothyroxinemia in iodine-deficient group (reduced FT4 with no significant increase in TSH) and hypothyroidism in 5ppm and 15ppm PTU-treated groups. The iodine-deficient and 15ppm PTU-treated groups had significantly lower serum FT3 and FT4 than the controls on PN14, PN21, and PN28 (P＜0.05). TSH levels were increased significantly in 5ppm and 15ppm PTU-treated offspring than controls (P＜0.05). On PN42, the concentrations of serum FT3, FT4, and TSH in iodine-deficient and PTU-treated groups were restored.
2、Alterations of body weights
Many studies have shown that PTU treatment reduces offspring body weights. Our data shown that the offspring's body weights in iodine-deficient,5ppm and 15ppm PTU-treated groups were statistically significant lower than those of controls on PN14, PN21, PN28, and PN42 (P＜0.05)
3、Impairment of neurons
Iodine deficiency and hypothyroidism reduce hippocampal surviving cells. To investigate whether or not hippocampal neuronal survival was impaired by iodine deficiency and hypothyroidism, histological examination of hippocampal neurons was performed on Nissl-stained sections. The results reveal lower surviving cells in the hippocampal neurons of offspring with low circulating TH levels in the CA1, CA3, and DG regions on PN21, PN28, and PN42(P＜0.05). The mean number of surviving cells in the hippocampus of the iodine-deficient,5 ppm PTU-treated, and 15 ppm PTU-treated rats was reduced compared to controls. On PN28 and PN42, compared with the control group, nerve fibers were found injured in the treated groups of regions CA3 and DG, as well as reduced in number. Injury appeared as fibers broken, disordered, bundled together, or fused with other fibers. In the control group, healthy nerve fibers were observed in all regions. The control nerve fibers were arranged in a good order with clear processes. Unlike regions CA3 and DG, nerve fibers in region CA1 were not yet damaged on PN28.
4、Effects on the related proteins
On PN14, PN21, PN28, and PN42, caveolin-1, synaptophysin, doublecortin and NCAM-180 were expressed in the CA1, CA3, and DG regions of the hippocampus of all groups, respectively. Significant upregulation of caveolin-1 was observed in rats exposed to the iodine-deficient,5 ppm PTU-adulterated, or 15 ppm PTU-adulterated diet relative to controls (P＜0.05). Synaptophysin was significantly down-regulated in rats exposed to the iodine-deficient,5 ppm PTU-adulterated, or 15 ppm PTU-adulterated diet relative to the controls (P＜0.05). A significant downregulation of doublecortin was observed in rats exposed to iodine-deficient and 15 ppm PTU-treated groups in CA1, CA3, and DG regions (P＜0.05). Upregulation of NCAM-180 in CA1, CA3, and DG regions were observed in iodine-deficient and 15 ppm PTU-treated rats from PN14 on (P＜0.05). Especially on PN42, we observed doublecortin decrease and NCAM-180 increase when TH levels were restored to a normal state, suggesting irreversible hippocampus impairment.
Conclusion
1、Iodine deficiency or PTU-induced hypothyroidism led to morphological damage in the hippocampus. Lower surviving cells in the hippocampal neurons of offspring with low circulating TH levels in the hippocampal subregions and nerve fibers were impaired.
2、Developmental iodine deficiency or PTU-induced hypothyroidism led to downregulation of doublecortin and synaptophysin and upregulation of Caveolin-1 and NCAM-180 in the hippocampal subregions at each time point.
3、The alterations of hippocampal morphology and expression of related proteins were still obvious on PN42 despite the recovery of TH to normal status.
引文
1 Kibirige MS, Hutchison S, Owen CJ, et al. Prevalence of maternal dietary iodine insufficiency in the north east of England:implications for the fetus. Arch Dis Child Fetal Neonatal Ed.2004; 89:F436-439.
2 陈祖培.对全民食盐加碘防治碘缺乏病的评价.国外医学.内分泌学分册.2002；22：268-271.
3 Bernal J. Action of thyroid hormone in brain. J Endocrinol Investig.2002; 25:268-288.
4 Berbel P, Obregon MJ, Bernal J, et al. Iodine supplementation during pregnancy:a public health challenge. Trends Endocrinol Metab.2007; 18(9):338-343.
5 Zoeller RT, Rover J. Timing of thyroid hormone action in the developing brain:Clinical observations and experimental findings. J Neuroendocrinol.2004; 16:809-818.
6 Morreale de Escobar G, Obregon MJ, Escobar del Rey F. Role of thyroid hormone during early brain development. Eur J Endocrinol.2004; 151:25-37.
7 Gilbert ME. Alterations in synaptic transmission and plasticity in area CA1 of adult hippocampus following developmental hypothyroidism. Dev Brain Res.2004; 148:11-18.
8 Mastorako G, Karoutsou El, Mizamtsidi M, et al. The menace of endocrine disruptors on thyroid hormone physiology and their impact on intrauterine developmet. Endocrine.2007; 31(3):219-237.
9 Morreale de Escobar G, Obregon MJ, Escobar del Rey F. Is neuropsychological development related to maternal hypothyroidism or to maternal hypothyroxinemia? J Clin Endocrinol Metab. 2000; 85:3975-3987.
10 Paxinos G, Watson C · The rat brain in stereotaxic coordinates-compact · Third edition · San Diego:Academic Press,1996.
11 Chan S, Kilby MD. Thyroid hormone and central nervous system development. Journal of Endocrinology.2000; 165:1-8.
12 Lazarus JH. Congenital hypothyroidism. Endocrinology.2005; 90:112-113.
13 Perez-Lopez FR. Iodine and thyroid hormones during pregnancy and postpartum. Gynecol Endocrinol.2007; 23(7):414-428.
14 Douglas F. The developing brain and maternal thyroid hormone:finding the links. Endocrinology.2004; 145(9):4034-4036.
15 Besedovsky H, Sorkin E. Network of immune-neuroendocrine interactions. Clin Exp Immunol. 1977; 27(1):1-12.
16 Rognoni JB, Lemarchand-Beraud T, Berthier C. Respective roles of circulating T4 and T3 in control of TSH secretion in severely iodide-deficient rats. Experientia.1984; 40(2):215-217.
17 Glinoer D. Clinical and biological consequences of iodine deficiency during pregnancy. Endocr Dev.2007; 10:62-85.
18 Sui L, Anderson WL, Gilbert ME. Impairment in short-term but enhanced long-term synaptic potentiation and ERK activation in adult hippocampus area CA1 following developmental thyroid hormone insufficiency. Toxicol Sci.2005; 85:647-656.
19 Nunez J, Celi FS, Ng L, et al. Multigenic control of thyroid hormone functions in the nervous system. Mol Cell Endocrinol.2008; 287(1-2):1-12.
20 Thompson CC, Potter GB. Thyroid hormone action in neural development. Cereb Cortex. 2000; 10:939-945.
21 Sala-Roca J, Estebanez-Perpina E, Balada F, et al. Effects of adult dysthyroidism on the morphology of hippocampal neurons. Behav Brain Res.2008; 188(2):348-354.
22 Kobayashi K, Tsuji R, Yoshioka T, et al. Effects of hypothyroidism induced by perinatal exposure to PTU on rat behavior and synaptic gene expression. Toxicology.2005; 212: 135-147.
23 Kim YO, Kim HJ, Kim GS, et al. Panax ginseng protects against global ischemia injury in rat hippocampus. J Med Food.2009; 12(1):71-76.
24陈祖培.当前碘缺乏病防治应注意的问题.中国地方病学杂志.2004；23(3)：193-194.
25 Kibirige MS, Hutchison S, Owen CJ, et al. Prevalence of maternal dietary iodine insuffieieney in the north east of England:implications for the fetus. Arch Dis Child Fetal Neonatal Ed.2004; 89:F436-439.
26 Anderson GW, Schoonover CM, Jones SA. Control of thyroid hormone action in the developing rat brain. Thyroid.2003; 13:1039-1056.
27 Bernal J, Guadano-Ferraz A, Morte B. Perspectives in the study of thyroid hormone in brain development and function. Thyroid.2003; 13:1005-1012.
28 Gilbert ME, Sui L. Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res. 2006; 1069:10-22.
29 Trushina E, Du-Charme J, Parisi J, et al. Neurological abnormalities in caveolin-1 knock out mice. Behav Brain Res.2006; 172:24-32.
30 Bu J, Bruckner SR, Sengoku T, et al. Glutamate regulates caveolin expression in rat hippocampal neurons. J Neurosci Res.2003; 72:185-190.
31 Schubert W, Frank PG, Razani B, et al. Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J Biol Chem.2001; 276:48619-48622.
32 Liu S, He Y, Dou Y, et al. Caveolin-1 restoration by cholesterol enhances the inhibitory effect of simvastatin on arginine vasopressin-induced cardiac fibroblasts proliferation. Mol Cell Biochem,2009, Doi:10.1007/s11010.009.0155.9.
33 Conrad PA, Smart EJ, Ying YS, et al. Caveolin cyclesbetween plasmamembrane caveolae and the Golgi complex by microtubule dependent and microtubule-independent steps. J Cell Biol. 1995; 131(6 Ptl):1421-1433.
34 Senou M, Costa M, Massart C, et al. Role of caveolin-1 in thyroid phenotype, cell homeostasis and hormone synthesis:"In vivo" study of caveolin-1 knockout mice. Am J Physiol Endocrinol Metab.2009; Doi:10.1152/ajpendo.90784.2008.
35 Couet J, Belanger MM, Roussel E, et al. Cell biology of caveolae and caveolin. Adv Drug Deliv Rev.2001; 49:223-235.
36 Krajewska WM, Maslowska I. Caveolins:structure and function in signal transduction. Cell Mol Biol Lett.2004; 9:195-220.
37 Tarsa L, Goda Y. Synaptophysin regulates activity-dependent synapse formation in cultured hippocampal neurons. PNAS.2002; 99:1012-1016.
38 Gomez M, Hernandez ML, Pazos MR, et al. Colocalization of CB1 receptors with L1 and GAP-43 in forebrain white matter regions during fetal rat brain development:evidence for a role of these receptors in axonal growth and guidance. Neuroscience.2008; 153:687-699.
39 Dzienis-Koronkiewicz E, Debek W, Chyczewski L. Use of synaptophysin immunohisto-chemistry in intestinal motility disorders. Eur J Pediatr Surg.2005; 15:392-398.
40 Scarfone E, Dememes D, Sans A. Synapsin I and Synaptophysin expression during ontogenesis of the mouse peripheral vestibular system. J Neurosci.1991; 11:1173-1181.
41 Liebau S, Vaida B, Storch A, et al. Maturation of synaptic contacts in differentiating neural stem cells. Stem Cells.2007; 25:1720-1729.
42 Ferreira A, Kosik KS, Greengard P, et al. Aberrant neurites and synaptic vesicle protein deficiency in synapsin Ⅱ-depleted neurons. Science.1994; 264(5161):977-979.
43 Chin LS, Li L, Ferreira A, et al. Impairment of axonao development and of aynaptogenesis in hippocampal neurons of synapsin Ⅰ-deficient mice. Pro Natl Acad Sci USA.1995; 92: 9230-9234.
44 Alzoubi KH, Alkadhi KA. A critical role of CREB in the impairment of late-phase LTP by adult onset hypothyroidism. Exp Neurol.2007; 203:63-71.
45 Dong J, Yin H, Liu W, et al. Congenital iodine deficiency and hypothyroidism impair LTP and decrease c-fos and c-jun expression in rat hippocampus. Neurotoxicology.2005; 26: 417-426.
46 Gerges NZ, Alkadhi KA. Hypothyroidism impairs late LTP in CA1 region but not in dentate gyrus of the intact rat hippocampus:MAPK involvement. Hippocampus.2004; 14:40-45.
47 Gilbert ME, Sui L, Walker MJ, et al. Thyroid hormone insufficiency during brain development reduces parvalbumin immunoreactivity and inhibitory function in the hippocampus. Endocrinology.2007; 148:92-102.
48 Navarro A, Anand-Apte B, Parat MO. A role for caveolae in cell migration. FASEB J.2004; 18:1801-1811.
49 Sarnat HB, Born DE. Synaptophysin immunocytochemistry with thermal intensification:a marker of terminal axonal maturation in the human fetal nervous system. Brain Dev.1999; 21: 41-50.
50 Newton IG, Forbes ME, Legault C, et al. Caloric restriction does not reverse aging-related changes in hippocampal BDNF. Neurobiol Aging.2005; 26:683-688.
51 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem.1976; 72:248-254.
52 Tang Z, Liu W, Yin H, et al. Investigation of intelligence quotient and psychomotor development in schoolchildren in areas with different degrees of iodine deficiency. Asia Pac J Clin Nutr.2007; 16:731-737.
53 Benton D, ILSI-Europe a.i.s.b.l. Micronutrient status, cognition and behavioral problems in childhood. Eur J Nutr.2008; 47:38-50.
54 Echarri A, Muriel O, Del-Pozo MA. Intracellular trafficking of raft/caveolae domains: insights from integrin signaling. Semin Cell Dev Biol.2007; 18:627-637.
55 Liu J, Wang XB, Park DS, et al. Caveolin-1 expression enhances endothelial capillary tubule formation. J Biol Chem.2002; 277:10661-10668.
56 Isshiki M, Ando J, Yamamoto K, et al. Sites of Ca (2+) wave initiation move with caveolae to the trailing edge of migrating cells. J Cell Sci.2002; 115(Pt 3):475-84.
57 Fielding CJ, Fielding PE. Relationship between cholesterol trafficking and signaling in rafts and caveolae. Biochim Biophys Acta.2003; 1610:219-228.
58 Murata M, Peranen J, Schreiner R, et al. VIP21/caveolin is a cholesterol-binding protein. Proc Natl Acad Sci USA.1995; 92:10339-10343.
59 Gaudreault SB, Blain JF, Gratton JP, et al. A role for caveolin-1 in post-injury reactive neuronal plasticity. J Neurochem.2005; 92:831-839.
60 Mauch DH, Nagler K, Schumacher S, et al. CNS synaptogenesis promoted by glia-derived cholesterol. Science.2001; 294:1354-1357.
61 Auso E, Lavado-Autric R, Cuevas E, et al. A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration. Endocrinology.2004; 145:4037-4047.
62 Farwell AP, Dubord-Tomasetti SA. Thyroid hormone regulates the expression of laminin in the developing rat cerebellum. Endocrinology.1999; 140:4221-4227.
63 Royland JE, Parker JS, Gilbert ME. A genomic analysis of subclinical hypothyroidism in hippocampus and neocortex of the developing rat brain. J Neuroendocrinol.2008; 20: 1319-1338.
64 Poirier J. Apolipoprotein E in animal models of CNS injury and in Alzheimer's disease. Trends Neurosci.1994; 17:525-530.
65 McMahon HT, Bolshakov VY, Janz R, et al. Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release. Proc Natl Acad Sci USA.1996; 93:4760-4764.
66 Fuentes-Santamaria V, Alvarado JC, Henkel CK, et al. Cochlear ablation in adult ferrets results in changes in insulin-like growth factor-1 and synaptophysin immunostaining in the cochlear nucleus. Neuroscience.2007; 148:1033-1047.
67 Fiala JC, Feinberg M, Popov V, et al. Synaptogenesis via dendritic filopodia in developing hippocampal area CA1.J Neurosci.1998; 18:8900-8911.
68 Nikizad H, Yon JH, Carter LB, et al. Early exposure to general anesthesia causes significant neuronal deletion in the developing rat brain. Ann N Y Acad Sci.2007; 1122:69-82.
69 Fielding PE, Fielding CJ. Plasma membrane caveolae mediate the efflux of cellular free cholesterol. Biochemistry.1995; 34:14288-14292.
70 Frank PG, Cheung MW, Pavlides S, et al. Caveolin-1 and regulation of cellular cholesterol homeostasis. Am J Physiol Heart Circ Physiol.2006; 291:H677-H686.
71 Zhou M, Parr RD, Petrescu AD, et al. Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Biochemistry.2004; 43:7288-7306.
72 Bernatchez PN, Bauer PM, Yu J, et al. Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides. Proc Natl Acad Sci USA.2005; 102: 761-766.
73 Taggart MJ, Leavis P, Feron O, et al. Inhibition of PKC alpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res.2000; 258:72-81.
74 Rehm H, Wiedenmann B, Betz H. Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane. EMBO J.1986; 5:535-541.
75 Braun JE, Madison DV. A novel SNAP25-caveolin complex correlates with the onset of persistent synaptic potentiation. J Neurosci.2000; 20:5997-6006.
76 Calloni GW, Penno CA, Cordova FM, et al. Congenital hypothyroidism alters the phosphorylation of ERKl/2 and p38MAPK in the hippocampus of neonatal rats. Brain Res Dev Brain Res.2005; 154(1):141-145.
77 Sui L, Gilbert ME. Pre-and postnatal propylthiouracil-induced hypothyroidism impairs synaptic transmission and plasticity in area CA1 of the neonatal rat hippocampus. Endocrinology.2003; 144:4195-4203.
78 Takeda A, Yamada K, Tamano H, et al. Hippocampal calcium dyshomeostasis and long-term potentiation in 2-week zinc deficiency. Neurochem Int.2008; 52:241-246.
79 Wang FD, Bian W, Kong LW, et al. Maternal zinc deficiency impairs brain nestin expression in prenatal and postnatal mice. Cell Res.2001; 11:135-141.
80 Koshibu K, Levitt P, Ahrens ET. Sex-specific, postpuberty changes in mouse brain structures revealed by three-dimensional magnetic resonance microscopy. Neuroimage.2004; 22(4): 1636-1645.
81 Lenroot RK, Giedd JN. Brain development in children and adolescents:insights from anatomical magnetic resonance imaging. Neurosci Biobehav Rev.2006; 30(6):718-729.
82 Aimone JB, Wiles J, Gage FH. Potential role for adult neurogenesis in the encoding of time in new memories. Nat Neurosci.2006; 9(6):723-727.
83 Farioli-Vecchioli S, Saraulli D, Costanzi M, et al. The timing of differentiation of adult hippocampal neurons is crucial for spatial memory. PLoS Biol.2008; 6(10):e246.
84 Eriksson PS, Perfilieva E, Bjork-Eriksson, et al. Neurogenesis in the adult human hippocampus. Nat Med.1998; 4:1313-1317.
85 Kuhn HG, Dickinson-Anson H, Gage FH. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci.1996; 16:2027-2033.
86 Prickaerts J, Koopmans G, Blockland A, et al. Leaining and adult neurogenesis:survival with or without proliferation? Neurobiol Learn Mem.2004; 81:1-11.
87 Sokolov EN, Nezlina NI. Long-term memory, neurogenesis, and signal novelty. Neurosci Behav Physiol.2004; 34:847-857.
88 Leuner B, Waddell J, Gould E, et al. Temporal discontiguity is neither necessary nor sufficient for learning-induced effects on adult neurogenesis. J Neurosci.2006; 26(52):13437-13442.
89 von Bohlen Und Halbach O. Immunohistological markers for staging neurogenesis in adult hippocampus. Cell Tissue Res.2007; 329(3):409-420.
90 Gleeson JG, Allen KM, Fox JW, et al. Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein. Cell. 1998,92(1):63-72.
91 Koizumi H, Higginbotham H, Poon T, et al. Doublecortin maintains bipolar shape and nuclear translocation during migration in the adult forebrain. Nat Neurosci.2006; 9(6): 779-786.
92 Kiss JZ, Troncoso E, Djebbara Z, et al. The role of neural cell adhesion molecules in plasticity and repair. Brain Res Brain Res Rev.2001; 36(2-3):175-184.
93 Walmod PS, Kolkovak, Berezin V, et al. Zippers make signals NCAM-mediated molecular interactions and signal transduction. Neurochem Res.2004; 29:2015-2035.
94 Bonfanti L. PSA-NCAM in mammalian structural plasticity and neurogenesis. Prog Neurobiol. 2006; 80(3):129-164.
95 Schuster T, Krug M, Hassan H, et al. Increase in proportion of hippocampal spine synapses expressing neural cell adhesion molecule NCAM180 following long-term potentiation. J Neurobiol.1998; 37(3):359-372.
96 Gdalyahu A, Ghosh I, Levy T, et al. DCX, a new mediator of the JNK pathway. EMBO J. 2004; 23(4):823-832.
97 Moores CA, Perderiset M, Kappeler C, et al. Distinct roles of doublecortin modulating the microtubule cytoskeleton. EMBO J.2006; 25(19):4448-4457.
98 Iglesias T, Caubin J, Stunnenberg HG, et al. Thyroid hormone-dependent transcriptional repression of neural cell adhesion molecule during brain maturation. EMBO J.1996; 15(16): 4307-4316.
99 Bernal J. Thyroid hormones and brain development. Vitam Horm.2005; 71:95-122.
100 Cuevas E, Auso E, Telefont M, et al. Transient maternal hypothyroxinemia at onset of corticogenesis alters tangential migration of MGE-derived neurons. Eur J Neurosei.2005; 22: 541-551.
101 Gould E, Tanapat P, Hastings NB, et al. Neurogenesis in adulthood:a possible role in learning. Trends Cogn Sci.1999; 3(5):186-192.
102 Joo JY, Kim BW, Lee JS, et al. Activation of NMDA receptors increases proliferation and differentiation of hippocampal neural progenitor cells. J Cell Sci.2007; 120(Pt 8):1358-1370.
103 Markakis EA. Gage FH. Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J Comp Neurol.1999; 406(4):449-460.
104 van Praag H, Schinder AF, Christie BR, et al. Functional neurogenesis in the adult hippocampus. Nature.2002; 415(6875):1030-1034.
105 Kempermann G, Wiskott L, Gage FH. Functional significance of adult neurogenesis. Curr Opin Neurobiol.2004; 14(2):186-191.
106 Kesner RP, Rogers J. An analysis of independence and interactions of brain substrates that subserve multiple attributes, memory systems, and underlying processes. Neurobiol Learn Mem. 2004; 82(3):199-215.
107 Treves A, Rolls ET. Computational analysis of the role of the hippocampus in memory. Hippocampus.1994; 4(3):374-391.
108 Ronn LC, Berezin V, Bock E. The neural cell adhesion molecule in synaptic plasticity and ageing. Int J Dev Neurosci.2000; 18(2-3):193-199.
109 Gilbert ME, Paczkowski C. Propylthiouracil (PTU)-induced hypothyroidism in the developing rat impairs synaptic transmission and plasticity in the dentate gyrus of the adult hippocampus. Brain Res Dev Brain Res.2003; 145(1):19-29.
1 Kibirige MS, Hutchison S, Owen CJ, et al. Prevalence of maternal dietary iodine insufficiency in the north east of England:implications for the fetus. Arch Dis Child Fetal Neonatal Ed.2004; 89:F436-439.
2 陈祖培.对全民食盐加碘防治碘缺乏病的评价.国外医学.内分泌学分册.2002；22：268-271.
3 Bernal J. Thyroid hormones and brain development. Vitam Horm.2005; 71:95-122.
4 Berbel P, Obregon MJ, Bernal J, et al. Iodine supplementation during pregnaney:a public health challenge. Trends Endocrinol Metab.2007; 18(9):338-343.
5 Zoeller RT, Rover J. Timing of thyroid hormone action in the developing brain:Clinical observations and experimental findings. J Neuroendocrinol.2004; 16:809-818.
6 Morreale de Escobar G, Obregon MJ, Escobar del Rey F. Role of thyroid hormone during early brain development. Eur J Endocrinol.2004; 151:25-37.
7 Morreale de Escobar G, Obregon MJ, Escobar del Rey F. Is neuropsychological development related to maternal hypothyroidism or to maternal hypothyroxinemia? J Clin Endocrinol Metab. 2000; 85:3975-3987.
8 Gilbert ME. Alterations in synaptic transmission and plasticity in area CA1 of adult hippocampus following developmental hypothyroidism. Dev Brain Res.2004; 148:11-18.
9 Mastorako G, Karoutsou El, Mizamtsidi M, et al. The menace of endocrine diruptors on thyroid hormone physiology and their impact on intrauterine developmet. Endocrine.2007; 31(3):219-237.
10 Rothberg KG, Heuser JE, Donzell WC, et al. Caveolin,a protein component of caveolae membrane coats.Cell.1992; 68(4):673-682.
11 Krajewska WM, Maslowska I. Caveolins:structure and function in signal transduction. Cell Mol Biol Lett.2004; 9 (2):195-220.
12 Couet J, Belanger MM, Roussel E, et al. Cell biology of caveolae and caveolin. Adv Drug Deliv Rev.2001; 49(3):223-235.
13 Bernatchez PN, Bauer PM, Yu J, et al. Dissecting the molecular control of endothelialNO synthase by caveolin-1 using cell-permeable peptides. Proc Natl Acad Sci USA.2005; 102(3): 761-766.
14 Epand RM, Sayer BG, Epand RF. Caveolin scaffolding region and cholesterol-rich domains inmembranes. J MolBiol.2005; 345(2):339-350.
15 Schubert W, Frank PG, Razani B, et al. Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J Biol Chem.2001; 276(52):48619-48622.
16 Minshall RD, Sessa WC, Stan RV, et al. Caveolin regulation of endothelial function. Am J Physiol Lung Cell Mol Physiol.2003; 285(6):L1179-L1183.
17 Frank PG, Woodman SE, Park DS, et al. Caveolin, caveolae, and endothelial cell function. Arterioscler Thromb Vasc Biol.2003; 23(7):1161-1168.
18 Razani B, Lisanti MP. Caveolin-deficient mice:insights into caveolar function human disease. J Clin Invest.2001; 108(11):1553-1561.
19 Senou M, Costa M, Massart C, et al. Role of caveolin-1 in thyroid phenotype, cell homeostasis and hormone synthesis:"In vivo" study of caveolin-1 knockout mice. Am J Physiol Endocrinol Metab.2009; Doi:10.1152/ajpendo.90784.2008.
20 Frank PG, Lee H, Park DS, et al. Genetic ablation of caveolin-1 confers protection against atherosclerosis. Arterioscler Thromb Vasc Biol.2004; 24(1):98-105.
21 Fu Y, Hoang A, Escher G, et al. Expression of caveolin-1 enhances cholesterol efflux in hepatic cells. J Biol Chem.2004; 279(14):14140-14146.
22 Conrad PA, Smart EJ, Ying YS, et al. Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule dependent and microtubule-independent steps. J Cell Biol.1995; 131(6 Pt1):1421-1433.
23 Taggart MJ, Leavis P, Feron O, et al. Inhibition of PKC alpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res.2000, 258:72-81.
24 Oka N, Yamamoto M, Schwencke C, et al. Caveolin interaction with protein kinase C. Isoenzyme-dependent regulation of kinase activity by the caveolin scaffolding domain peptide. J Biol Chem.1997; 272(52):33416-33421.
25 Bhatnagar A, Sheffler DJ, Kroeze WK, et al. Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Galphaq-coupled protein receptors. J Biol Chem.2004; 279(33):34614-34623.
26 Alzoubi KH, Alkadhi KA. A critical role of CREB in the impairment of late-phase LTP by adult onset hypothyroidism. Exp Neurol.2007; 203:63-71.
27 Dong J, Yin H, Liu W, et al. Congenital iodine deficiency and hypothyroidism impair LTP and decrease c-fos and c-jun expression in rat hippocampus. Neurotoxicology.2005; 26: 417-426.
28 Gerges NZ, Alkadhi KA. Hypothyroidism impairs late LTP in CA1 region but not in dentate gyrus of the intact rat hippocampus:MAPK involvement. Hippocampus.2004; 14:40-45.
29 Gilbert ME, Sui L, Walker MJ, et al. Thyroid hormone insufficiency during brain development reduces parvalbumin immunoreactivity and inhibitory function in the hippocampus. Endocrinology.2007; 148:92-102.
30 Sui L, Anderson WL, Gilbert ME. Impairment in short-term but enhanced long-term synaptic potentiation and ERK activation in adult hippocampus area CA1 following developmental thyroid hormone insufficiency. Toxicol Sci.2005; 85:647-656.
31 Navarro A, Anand-Apte B, Parat MO. A role for caveolae in cell migration. FASEB J.2004; 18:1801-1811.
32 von Bohlen Und Halbach O. Immunohistological markers for staging neurogenesis in adult hippocampus. Cell Tissue Res.2007; 329(3):409-420.
33 Gleeson JG, Allen KM, Fox JW, et al. Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein. Cell. 1998; 92(1):63-72.
34 Koizumi H, Higginbotham H, Poon T, et al. Doublecortin maintains bipolar shape and nuclear translocation during migration in the adult forebrain. Nat Neurosci.2006; 9(6): 779-786.
35 Koshibu K, Levitt P, Ahrens ET. Sex-specific, postpuberty changes in mouse brain structures revealed by three-dimensional magnetic resonance microscopy. Neuroimage.2004; 22(4): 1636-1645.
36 Lenroot RK, Giedd JN. Brain development in children and adolescents:insights from anatomical magnetic resonance imaging. Neurosci Biobehav Rev.2006; 30(6):718-729.
37 Aimone JB, Wiles J, Gage FH. Potential role for adult neurogenesis in the encoding of time in new memories. Nat Neurosci.2006; 9(6):723-727.
38 Farioli-Vecchioli S, Saraulli D, Costanzi M, et al. The timing of differentiation of adult hippocampal neurons is crucial for spatial memory. PLoS Biol.2008; 6(10):e246.
39 Prickaerts J, Koopmans G, Blockland A, et al. Learning and adult neurogenesis:survival with or without proliferation? Neurobiol Learn Mem.2004; 81:1-11.
40 Sokolov EN, Nezlina NI. Long-term memory, neurogenesis, and signal novelty. Neurosci Behav Physiol.2004; 34:847-857.
41 Sala-Roca J, Estebanez-Perpina E, Balada F, et al. Effects of adult dysthyroidism on the morphology of hippocampal neurons. Behav Brain Res.2008; 188(2):348-354.
42 Prickaerts J, Koopmans G, Blokland A, et al. Learning and adult neurogenesis:survival with or without proliferation? Neurobiol Learn Mem.2004; 81(1):1-11.
43 Gould E, Tanapat P, Hastings NB, et al. Neurogenesis in adulthood:a possible role in learning. Trends Cogn Sci.1999; 3(5):186-192.
44 Joo JY, Kim BW, Lee JS, et al. Activation of NMDA receptors increases proliferation and differentiation of hippocampal neural progenitor cells. J Cell Sci.2007; 120(Pt 8):1358-1370.
45 Markakis EA, Gage FH. Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J Comp Neurol.1999; 406(4):449-460.
46 van Praag H, Schinder AF, Christie BR, et al. Functional neurogenesis in the adult hippocampus. Nature.2002; 415(6875):1030-1034.
47 Kempermann G, Wiskott L, Gage FH. Functional significance of adult neurogenesis. Curr Opin Neurobiol.2004; 14(2):186-191.
48 Kesner RP, Rogers J. An analysis of independence and interactions of brain substrates that subserve multiple attributes, memory systems, and underlying processes. Neurobiol Learn Mem. 2004; 82(3):199-215.
49 Treves A, Rolls ET. Computational analysis of the role of the hippocampus in memory. Hippocampus.1994; 4(3):374-391.
50 Leuner B, Waddell J, Gould E, et al. Temporal discontiguity is neither necessary nor sufficient for learning-induced effects on adult neurogenesis. J Neurosci.2006; 26(52):13437-13442.
51 Takeda A, Yamada K, Tamano H, et al. Hippocampal calcium dyshomeostasis and long-term potentiation in 2-week zinc deficiency. Neurochem Int.2008; 52:241-246.
52 Wang FD, Bian W, Kong LW, et al. maternal zinc deficiency impairs brain nestin expression in prenatal and postnatal mice. Cell Res.2001; 11:135-141.
53 Gdalyahu A, Ghosh I, Levy T, et al. DCX, a new mediator of the JNK pathway. EMBO J. 2004; 23(4):823-832.
54 Moores CA, Perderiset M, Kappeler C, et al. Distinct roles of doublecortin modulating the microtubule cytoskeleton. EMBO J.2006; 25(19):4448-4457.
55 Royland JE, Parker JS, Gilbert ME. A genomic analysis of subclinical hypothyroidism in hippocampus and neocortex of the developing rat brain. J Neuroendocrinol.2008; 20(12): 1319-1338.
56 Doberty P, Walsh FS. CAM-FGF receptor interations:a model for axonal growth. Mol Cell Neurosci.1996; 8(1):99-111.
57 Schuster T, Krug M, Hassan H, et al. Increase in proportion of hippocampal spine synapses expressing neural cell adhesion molecule NCAM180 following long-term potentiation. J Neurobiol.1998; 37(3):359-372.
58 Kasper C, Rasmusen H, Kastrup JS, et al. Structural basis of cell-cell adhesion by NCAM. Nature Structural Biol.2000; 7:387-393.
59 Eckhardt M, Bukalo O, Chazal G, et al. Mice deficient in the polysialyltransferase ST8SiaⅣ/PST-1 allow discrimination of the roles of neural cell adhesion molecule protein and polysialic acid in neural development and synaptic plasticity. J Neurosci.2000; 20(14): 5234-5244.
60 Walmod PS, Kolkovak, Berezin V, et al. Zippers make signals NCAM-mediated molecular interactions and signal transduction. Neurochem Res.2004; 29(11):2015-2035.
61 Auso E, Lavado-Autric R, Cuevas E, et al. A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration. Endocrinology.2004; 145(9):4037-4047.
62 Farwell AP, Dubord-Tomasetti SA. Thyroid hormone regulates the expression of laminin in the developing rat cerebellum. Endocrinology.1999; 140(9):4221-4227.
63 Ronn LC, Berezin V, Bock E. The neural cell adhesion molecule in synaptic plasticity and ageing. Int J Dev Neurosci.2000; 18(2-3):193-199.
64 Iglesias T, Caubin J, Stunnenberg HG, et al. Thyroid hormone-dependent transcriptional repression of neural cell adhesion molecule during brain maturation. EMBO J.1996; 15(16): 4307-4316.
65 Tarsa L, Goda Y. Synaptophysin regulates activity-dependent synapse formation in cultured hippocampal neurons. PNAS.2002; 99(2):1012-1016.
66 Gomez M, Hernandez ML, Pazos MR, et al. Colocalization of CB1 receptors with L1 and GAP-43 in forebrain white matter regions during fetal rat brain development:evidence for a role of these receptors in axonal growth and guidance. Neuroscience.2008; 153:687-699.
67 Dzienis-Koronkiewicz E, Debek W, Chyczewski L. Use of synaptophysin immunohistochemistry in intestinal motility disorders. Eur J Pediatr Surg.2005; 15 (6): 392-398.
68 Scarfone E, Dememes D, Sans A. Synapsin I and Synaptophysin expression during ontogenesis of the mouse peripheral vestibular system. J Neurosci.1991; 11:1173-1181.
69 Liebau S, Vaida B, Storch A, et al. Maturation of synaptic contacts in differentiating neural stem cells. Stem Cells.2007; 25:1720-1729.
70 Ferreira A, Kosik KS, Greengard P, et al. Aberrant neurites and synaptic vesicle protein deficiency in synapsin Ⅱ-depleted neurons. Science.1994; 264(5161):977-979.
71 Chin LS, Li L, Ferreira A, et al. Impairment of axonao development and of aynaptogenesis in hippocampal neurons of synapsin I-deficient mice. Pro Natl Acad Sci USA.1995; 92: 9230-9234
72 Angers A, Fioravante D, Chin J, et al. Serotonin stimulates phosphorylation of aplysia synapsin and alters its subcellular distribution in sensory neurons. J Neurosci.2002; 22(13): 5412-5422.
73 Fiala JC, Feinberg M, Popov V, et al. Synaptogenesis via dendritic filopodia in developing hippocampal area CA1. J Neurosci.1998; 18:8900-8911.
74 Nikizad H, Yon JH, Carter LB, et al. Early exposure to general anesthesia causes significant neuronal deletion in the developing rat brain. Ann N Y Acad Sci.2007; 1122:69-82.
75 Bernal J. Action of thyroid hormone in brain. J Endocrinol Invest.2002; 25:268-288.
76 Dong H, Yauk CL, Rowan-Carroll A, et al. Identification of thyroid hormone receptor binding sites and target genes using ChIP-on-chip in developing mouse cerebellum. PLoS One.2009; 4(2):e4610.
2 陈祖培.对全民食盐加碘防治碘缺乏病的评价.国外医学.内分泌学分册.2002；22：268-271.
3 Bernal J. Action of thyroid hormone in brain. J Endocrinol Investig.2002; 25:268-288.
4 Berbel P, Obregon MJ, Bernal J, et al. Iodine supplementation during pregnancy:a public health challenge. Trends Endocrinol Metab.2007; 18(9):338-343.
5 Zoeller RT, Rover J. Timing of thyroid hormone action in the developing brain:Clinical observations and experimental findings. J Neuroendocrinol.2004; 16:809-818.
6 Morreale de Escobar G, Obregon MJ, Escobar del Rey F. Role of thyroid hormone during early brain development. Eur J Endocrinol.2004; 151:25-37.
7 Gilbert ME. Alterations in synaptic transmission and plasticity in area CA1 of adult hippocampus following developmental hypothyroidism. Dev Brain Res.2004; 148:11-18.
8 Mastorako G, Karoutsou El, Mizamtsidi M, et al. The menace of endocrine disruptors on thyroid hormone physiology and their impact on intrauterine developmet. Endocrine.2007; 31(3):219-237.
9 Morreale de Escobar G, Obregon MJ, Escobar del Rey F. Is neuropsychological development related to maternal hypothyroidism or to maternal hypothyroxinemia? J Clin Endocrinol Metab. 2000; 85:3975-3987.
10 Paxinos G, Watson C · The rat brain in stereotaxic coordinates-compact · Third edition · San Diego:Academic Press,1996.
11 Chan S, Kilby MD. Thyroid hormone and central nervous system development. Journal of Endocrinology.2000; 165:1-8.
12 Lazarus JH. Congenital hypothyroidism. Endocrinology.2005; 90:112-113.
13 Perez-Lopez FR. Iodine and thyroid hormones during pregnancy and postpartum. Gynecol Endocrinol.2007; 23(7):414-428.
14 Douglas F. The developing brain and maternal thyroid hormone:finding the links. Endocrinology.2004; 145(9):4034-4036.
15 Besedovsky H, Sorkin E. Network of immune-neuroendocrine interactions. Clin Exp Immunol. 1977; 27(1):1-12.
16 Rognoni JB, Lemarchand-Beraud T, Berthier C. Respective roles of circulating T4 and T3 in control of TSH secretion in severely iodide-deficient rats. Experientia.1984; 40(2):215-217.
17 Glinoer D. Clinical and biological consequences of iodine deficiency during pregnancy. Endocr Dev.2007; 10:62-85.
18 Sui L, Anderson WL, Gilbert ME. Impairment in short-term but enhanced long-term synaptic potentiation and ERK activation in adult hippocampus area CA1 following developmental thyroid hormone insufficiency. Toxicol Sci.2005; 85:647-656.
19 Nunez J, Celi FS, Ng L, et al. Multigenic control of thyroid hormone functions in the nervous system. Mol Cell Endocrinol.2008; 287(1-2):1-12.
20 Thompson CC, Potter GB. Thyroid hormone action in neural development. Cereb Cortex. 2000; 10:939-945.
21 Sala-Roca J, Estebanez-Perpina E, Balada F, et al. Effects of adult dysthyroidism on the morphology of hippocampal neurons. Behav Brain Res.2008; 188(2):348-354.
22 Kobayashi K, Tsuji R, Yoshioka T, et al. Effects of hypothyroidism induced by perinatal exposure to PTU on rat behavior and synaptic gene expression. Toxicology.2005; 212: 135-147.
23 Kim YO, Kim HJ, Kim GS, et al. Panax ginseng protects against global ischemia injury in rat hippocampus. J Med Food.2009; 12(1):71-76.
24陈祖培.当前碘缺乏病防治应注意的问题.中国地方病学杂志.2004；23(3)：193-194.
25 Kibirige MS, Hutchison S, Owen CJ, et al. Prevalence of maternal dietary iodine insuffieieney in the north east of England:implications for the fetus. Arch Dis Child Fetal Neonatal Ed.2004; 89:F436-439.
26 Anderson GW, Schoonover CM, Jones SA. Control of thyroid hormone action in the developing rat brain. Thyroid.2003; 13:1039-1056.
27 Bernal J, Guadano-Ferraz A, Morte B. Perspectives in the study of thyroid hormone in brain development and function. Thyroid.2003; 13:1005-1012.
28 Gilbert ME, Sui L. Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res. 2006; 1069:10-22.
29 Trushina E, Du-Charme J, Parisi J, et al. Neurological abnormalities in caveolin-1 knock out mice. Behav Brain Res.2006; 172:24-32.
30 Bu J, Bruckner SR, Sengoku T, et al. Glutamate regulates caveolin expression in rat hippocampal neurons. J Neurosci Res.2003; 72:185-190.
31 Schubert W, Frank PG, Razani B, et al. Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J Biol Chem.2001; 276:48619-48622.
32 Liu S, He Y, Dou Y, et al. Caveolin-1 restoration by cholesterol enhances the inhibitory effect of simvastatin on arginine vasopressin-induced cardiac fibroblasts proliferation. Mol Cell Biochem,2009, Doi:10.1007/s11010.009.0155.9.
33 Conrad PA, Smart EJ, Ying YS, et al. Caveolin cyclesbetween plasmamembrane caveolae and the Golgi complex by microtubule dependent and microtubule-independent steps. J Cell Biol. 1995; 131(6 Ptl):1421-1433.
34 Senou M, Costa M, Massart C, et al. Role of caveolin-1 in thyroid phenotype, cell homeostasis and hormone synthesis:"In vivo" study of caveolin-1 knockout mice. Am J Physiol Endocrinol Metab.2009; Doi:10.1152/ajpendo.90784.2008.
35 Couet J, Belanger MM, Roussel E, et al. Cell biology of caveolae and caveolin. Adv Drug Deliv Rev.2001; 49:223-235.
36 Krajewska WM, Maslowska I. Caveolins:structure and function in signal transduction. Cell Mol Biol Lett.2004; 9:195-220.
37 Tarsa L, Goda Y. Synaptophysin regulates activity-dependent synapse formation in cultured hippocampal neurons. PNAS.2002; 99:1012-1016.
38 Gomez M, Hernandez ML, Pazos MR, et al. Colocalization of CB1 receptors with L1 and GAP-43 in forebrain white matter regions during fetal rat brain development:evidence for a role of these receptors in axonal growth and guidance. Neuroscience.2008; 153:687-699.
39 Dzienis-Koronkiewicz E, Debek W, Chyczewski L. Use of synaptophysin immunohisto-chemistry in intestinal motility disorders. Eur J Pediatr Surg.2005; 15:392-398.
40 Scarfone E, Dememes D, Sans A. Synapsin I and Synaptophysin expression during ontogenesis of the mouse peripheral vestibular system. J Neurosci.1991; 11:1173-1181.
41 Liebau S, Vaida B, Storch A, et al. Maturation of synaptic contacts in differentiating neural stem cells. Stem Cells.2007; 25:1720-1729.
42 Ferreira A, Kosik KS, Greengard P, et al. Aberrant neurites and synaptic vesicle protein deficiency in synapsin Ⅱ-depleted neurons. Science.1994; 264(5161):977-979.
43 Chin LS, Li L, Ferreira A, et al. Impairment of axonao development and of aynaptogenesis in hippocampal neurons of synapsin Ⅰ-deficient mice. Pro Natl Acad Sci USA.1995; 92: 9230-9234.
44 Alzoubi KH, Alkadhi KA. A critical role of CREB in the impairment of late-phase LTP by adult onset hypothyroidism. Exp Neurol.2007; 203:63-71.
45 Dong J, Yin H, Liu W, et al. Congenital iodine deficiency and hypothyroidism impair LTP and decrease c-fos and c-jun expression in rat hippocampus. Neurotoxicology.2005; 26: 417-426.
46 Gerges NZ, Alkadhi KA. Hypothyroidism impairs late LTP in CA1 region but not in dentate gyrus of the intact rat hippocampus:MAPK involvement. Hippocampus.2004; 14:40-45.
47 Gilbert ME, Sui L, Walker MJ, et al. Thyroid hormone insufficiency during brain development reduces parvalbumin immunoreactivity and inhibitory function in the hippocampus. Endocrinology.2007; 148:92-102.
48 Navarro A, Anand-Apte B, Parat MO. A role for caveolae in cell migration. FASEB J.2004; 18:1801-1811.
49 Sarnat HB, Born DE. Synaptophysin immunocytochemistry with thermal intensification:a marker of terminal axonal maturation in the human fetal nervous system. Brain Dev.1999; 21: 41-50.
50 Newton IG, Forbes ME, Legault C, et al. Caloric restriction does not reverse aging-related changes in hippocampal BDNF. Neurobiol Aging.2005; 26:683-688.
51 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem.1976; 72:248-254.
52 Tang Z, Liu W, Yin H, et al. Investigation of intelligence quotient and psychomotor development in schoolchildren in areas with different degrees of iodine deficiency. Asia Pac J Clin Nutr.2007; 16:731-737.
53 Benton D, ILSI-Europe a.i.s.b.l. Micronutrient status, cognition and behavioral problems in childhood. Eur J Nutr.2008; 47:38-50.
54 Echarri A, Muriel O, Del-Pozo MA. Intracellular trafficking of raft/caveolae domains: insights from integrin signaling. Semin Cell Dev Biol.2007; 18:627-637.
55 Liu J, Wang XB, Park DS, et al. Caveolin-1 expression enhances endothelial capillary tubule formation. J Biol Chem.2002; 277:10661-10668.
56 Isshiki M, Ando J, Yamamoto K, et al. Sites of Ca (2+) wave initiation move with caveolae to the trailing edge of migrating cells. J Cell Sci.2002; 115(Pt 3):475-84.
57 Fielding CJ, Fielding PE. Relationship between cholesterol trafficking and signaling in rafts and caveolae. Biochim Biophys Acta.2003; 1610:219-228.
58 Murata M, Peranen J, Schreiner R, et al. VIP21/caveolin is a cholesterol-binding protein. Proc Natl Acad Sci USA.1995; 92:10339-10343.
59 Gaudreault SB, Blain JF, Gratton JP, et al. A role for caveolin-1 in post-injury reactive neuronal plasticity. J Neurochem.2005; 92:831-839.
60 Mauch DH, Nagler K, Schumacher S, et al. CNS synaptogenesis promoted by glia-derived cholesterol. Science.2001; 294:1354-1357.
61 Auso E, Lavado-Autric R, Cuevas E, et al. A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration. Endocrinology.2004; 145:4037-4047.
62 Farwell AP, Dubord-Tomasetti SA. Thyroid hormone regulates the expression of laminin in the developing rat cerebellum. Endocrinology.1999; 140:4221-4227.
63 Royland JE, Parker JS, Gilbert ME. A genomic analysis of subclinical hypothyroidism in hippocampus and neocortex of the developing rat brain. J Neuroendocrinol.2008; 20: 1319-1338.
64 Poirier J. Apolipoprotein E in animal models of CNS injury and in Alzheimer's disease. Trends Neurosci.1994; 17:525-530.
65 McMahon HT, Bolshakov VY, Janz R, et al. Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release. Proc Natl Acad Sci USA.1996; 93:4760-4764.
66 Fuentes-Santamaria V, Alvarado JC, Henkel CK, et al. Cochlear ablation in adult ferrets results in changes in insulin-like growth factor-1 and synaptophysin immunostaining in the cochlear nucleus. Neuroscience.2007; 148:1033-1047.
67 Fiala JC, Feinberg M, Popov V, et al. Synaptogenesis via dendritic filopodia in developing hippocampal area CA1.J Neurosci.1998; 18:8900-8911.
68 Nikizad H, Yon JH, Carter LB, et al. Early exposure to general anesthesia causes significant neuronal deletion in the developing rat brain. Ann N Y Acad Sci.2007; 1122:69-82.
69 Fielding PE, Fielding CJ. Plasma membrane caveolae mediate the efflux of cellular free cholesterol. Biochemistry.1995; 34:14288-14292.
70 Frank PG, Cheung MW, Pavlides S, et al. Caveolin-1 and regulation of cellular cholesterol homeostasis. Am J Physiol Heart Circ Physiol.2006; 291:H677-H686.
71 Zhou M, Parr RD, Petrescu AD, et al. Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo. Biochemistry.2004; 43:7288-7306.
72 Bernatchez PN, Bauer PM, Yu J, et al. Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides. Proc Natl Acad Sci USA.2005; 102: 761-766.
73 Taggart MJ, Leavis P, Feron O, et al. Inhibition of PKC alpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res.2000; 258:72-81.
74 Rehm H, Wiedenmann B, Betz H. Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane. EMBO J.1986; 5:535-541.
75 Braun JE, Madison DV. A novel SNAP25-caveolin complex correlates with the onset of persistent synaptic potentiation. J Neurosci.2000; 20:5997-6006.
76 Calloni GW, Penno CA, Cordova FM, et al. Congenital hypothyroidism alters the phosphorylation of ERKl/2 and p38MAPK in the hippocampus of neonatal rats. Brain Res Dev Brain Res.2005; 154(1):141-145.
77 Sui L, Gilbert ME. Pre-and postnatal propylthiouracil-induced hypothyroidism impairs synaptic transmission and plasticity in area CA1 of the neonatal rat hippocampus. Endocrinology.2003; 144:4195-4203.
78 Takeda A, Yamada K, Tamano H, et al. Hippocampal calcium dyshomeostasis and long-term potentiation in 2-week zinc deficiency. Neurochem Int.2008; 52:241-246.
79 Wang FD, Bian W, Kong LW, et al. Maternal zinc deficiency impairs brain nestin expression in prenatal and postnatal mice. Cell Res.2001; 11:135-141.
80 Koshibu K, Levitt P, Ahrens ET. Sex-specific, postpuberty changes in mouse brain structures revealed by three-dimensional magnetic resonance microscopy. Neuroimage.2004; 22(4): 1636-1645.
81 Lenroot RK, Giedd JN. Brain development in children and adolescents:insights from anatomical magnetic resonance imaging. Neurosci Biobehav Rev.2006; 30(6):718-729.
82 Aimone JB, Wiles J, Gage FH. Potential role for adult neurogenesis in the encoding of time in new memories. Nat Neurosci.2006; 9(6):723-727.
83 Farioli-Vecchioli S, Saraulli D, Costanzi M, et al. The timing of differentiation of adult hippocampal neurons is crucial for spatial memory. PLoS Biol.2008; 6(10):e246.
84 Eriksson PS, Perfilieva E, Bjork-Eriksson, et al. Neurogenesis in the adult human hippocampus. Nat Med.1998; 4:1313-1317.
85 Kuhn HG, Dickinson-Anson H, Gage FH. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci.1996; 16:2027-2033.
86 Prickaerts J, Koopmans G, Blockland A, et al. Leaining and adult neurogenesis:survival with or without proliferation? Neurobiol Learn Mem.2004; 81:1-11.
87 Sokolov EN, Nezlina NI. Long-term memory, neurogenesis, and signal novelty. Neurosci Behav Physiol.2004; 34:847-857.
88 Leuner B, Waddell J, Gould E, et al. Temporal discontiguity is neither necessary nor sufficient for learning-induced effects on adult neurogenesis. J Neurosci.2006; 26(52):13437-13442.
89 von Bohlen Und Halbach O. Immunohistological markers for staging neurogenesis in adult hippocampus. Cell Tissue Res.2007; 329(3):409-420.
90 Gleeson JG, Allen KM, Fox JW, et al. Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein. Cell. 1998,92(1):63-72.
91 Koizumi H, Higginbotham H, Poon T, et al. Doublecortin maintains bipolar shape and nuclear translocation during migration in the adult forebrain. Nat Neurosci.2006; 9(6): 779-786.
92 Kiss JZ, Troncoso E, Djebbara Z, et al. The role of neural cell adhesion molecules in plasticity and repair. Brain Res Brain Res Rev.2001; 36(2-3):175-184.
93 Walmod PS, Kolkovak, Berezin V, et al. Zippers make signals NCAM-mediated molecular interactions and signal transduction. Neurochem Res.2004; 29:2015-2035.
94 Bonfanti L. PSA-NCAM in mammalian structural plasticity and neurogenesis. Prog Neurobiol. 2006; 80(3):129-164.
95 Schuster T, Krug M, Hassan H, et al. Increase in proportion of hippocampal spine synapses expressing neural cell adhesion molecule NCAM180 following long-term potentiation. J Neurobiol.1998; 37(3):359-372.
96 Gdalyahu A, Ghosh I, Levy T, et al. DCX, a new mediator of the JNK pathway. EMBO J. 2004; 23(4):823-832.
97 Moores CA, Perderiset M, Kappeler C, et al. Distinct roles of doublecortin modulating the microtubule cytoskeleton. EMBO J.2006; 25(19):4448-4457.
98 Iglesias T, Caubin J, Stunnenberg HG, et al. Thyroid hormone-dependent transcriptional repression of neural cell adhesion molecule during brain maturation. EMBO J.1996; 15(16): 4307-4316.
99 Bernal J. Thyroid hormones and brain development. Vitam Horm.2005; 71:95-122.
100 Cuevas E, Auso E, Telefont M, et al. Transient maternal hypothyroxinemia at onset of corticogenesis alters tangential migration of MGE-derived neurons. Eur J Neurosei.2005; 22: 541-551.
101 Gould E, Tanapat P, Hastings NB, et al. Neurogenesis in adulthood:a possible role in learning. Trends Cogn Sci.1999; 3(5):186-192.
102 Joo JY, Kim BW, Lee JS, et al. Activation of NMDA receptors increases proliferation and differentiation of hippocampal neural progenitor cells. J Cell Sci.2007; 120(Pt 8):1358-1370.
103 Markakis EA. Gage FH. Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J Comp Neurol.1999; 406(4):449-460.
104 van Praag H, Schinder AF, Christie BR, et al. Functional neurogenesis in the adult hippocampus. Nature.2002; 415(6875):1030-1034.
105 Kempermann G, Wiskott L, Gage FH. Functional significance of adult neurogenesis. Curr Opin Neurobiol.2004; 14(2):186-191.
106 Kesner RP, Rogers J. An analysis of independence and interactions of brain substrates that subserve multiple attributes, memory systems, and underlying processes. Neurobiol Learn Mem. 2004; 82(3):199-215.
107 Treves A, Rolls ET. Computational analysis of the role of the hippocampus in memory. Hippocampus.1994; 4(3):374-391.
108 Ronn LC, Berezin V, Bock E. The neural cell adhesion molecule in synaptic plasticity and ageing. Int J Dev Neurosci.2000; 18(2-3):193-199.
109 Gilbert ME, Paczkowski C. Propylthiouracil (PTU)-induced hypothyroidism in the developing rat impairs synaptic transmission and plasticity in the dentate gyrus of the adult hippocampus. Brain Res Dev Brain Res.2003; 145(1):19-29.
1 Kibirige MS, Hutchison S, Owen CJ, et al. Prevalence of maternal dietary iodine insufficiency in the north east of England:implications for the fetus. Arch Dis Child Fetal Neonatal Ed.2004; 89:F436-439.
2 陈祖培.对全民食盐加碘防治碘缺乏病的评价.国外医学.内分泌学分册.2002；22：268-271.
3 Bernal J. Thyroid hormones and brain development. Vitam Horm.2005; 71:95-122.
4 Berbel P, Obregon MJ, Bernal J, et al. Iodine supplementation during pregnaney:a public health challenge. Trends Endocrinol Metab.2007; 18(9):338-343.
5 Zoeller RT, Rover J. Timing of thyroid hormone action in the developing brain:Clinical observations and experimental findings. J Neuroendocrinol.2004; 16:809-818.
6 Morreale de Escobar G, Obregon MJ, Escobar del Rey F. Role of thyroid hormone during early brain development. Eur J Endocrinol.2004; 151:25-37.
7 Morreale de Escobar G, Obregon MJ, Escobar del Rey F. Is neuropsychological development related to maternal hypothyroidism or to maternal hypothyroxinemia? J Clin Endocrinol Metab. 2000; 85:3975-3987.
8 Gilbert ME. Alterations in synaptic transmission and plasticity in area CA1 of adult hippocampus following developmental hypothyroidism. Dev Brain Res.2004; 148:11-18.
9 Mastorako G, Karoutsou El, Mizamtsidi M, et al. The menace of endocrine diruptors on thyroid hormone physiology and their impact on intrauterine developmet. Endocrine.2007; 31(3):219-237.
10 Rothberg KG, Heuser JE, Donzell WC, et al. Caveolin,a protein component of caveolae membrane coats.Cell.1992; 68(4):673-682.
11 Krajewska WM, Maslowska I. Caveolins:structure and function in signal transduction. Cell Mol Biol Lett.2004; 9 (2):195-220.
12 Couet J, Belanger MM, Roussel E, et al. Cell biology of caveolae and caveolin. Adv Drug Deliv Rev.2001; 49(3):223-235.
13 Bernatchez PN, Bauer PM, Yu J, et al. Dissecting the molecular control of endothelialNO synthase by caveolin-1 using cell-permeable peptides. Proc Natl Acad Sci USA.2005; 102(3): 761-766.
14 Epand RM, Sayer BG, Epand RF. Caveolin scaffolding region and cholesterol-rich domains inmembranes. J MolBiol.2005; 345(2):339-350.
15 Schubert W, Frank PG, Razani B, et al. Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J Biol Chem.2001; 276(52):48619-48622.
16 Minshall RD, Sessa WC, Stan RV, et al. Caveolin regulation of endothelial function. Am J Physiol Lung Cell Mol Physiol.2003; 285(6):L1179-L1183.
17 Frank PG, Woodman SE, Park DS, et al. Caveolin, caveolae, and endothelial cell function. Arterioscler Thromb Vasc Biol.2003; 23(7):1161-1168.
18 Razani B, Lisanti MP. Caveolin-deficient mice:insights into caveolar function human disease. J Clin Invest.2001; 108(11):1553-1561.
19 Senou M, Costa M, Massart C, et al. Role of caveolin-1 in thyroid phenotype, cell homeostasis and hormone synthesis:"In vivo" study of caveolin-1 knockout mice. Am J Physiol Endocrinol Metab.2009; Doi:10.1152/ajpendo.90784.2008.
20 Frank PG, Lee H, Park DS, et al. Genetic ablation of caveolin-1 confers protection against atherosclerosis. Arterioscler Thromb Vasc Biol.2004; 24(1):98-105.
21 Fu Y, Hoang A, Escher G, et al. Expression of caveolin-1 enhances cholesterol efflux in hepatic cells. J Biol Chem.2004; 279(14):14140-14146.
22 Conrad PA, Smart EJ, Ying YS, et al. Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule dependent and microtubule-independent steps. J Cell Biol.1995; 131(6 Pt1):1421-1433.
23 Taggart MJ, Leavis P, Feron O, et al. Inhibition of PKC alpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res.2000, 258:72-81.
24 Oka N, Yamamoto M, Schwencke C, et al. Caveolin interaction with protein kinase C. Isoenzyme-dependent regulation of kinase activity by the caveolin scaffolding domain peptide. J Biol Chem.1997; 272(52):33416-33421.
25 Bhatnagar A, Sheffler DJ, Kroeze WK, et al. Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Galphaq-coupled protein receptors. J Biol Chem.2004; 279(33):34614-34623.
26 Alzoubi KH, Alkadhi KA. A critical role of CREB in the impairment of late-phase LTP by adult onset hypothyroidism. Exp Neurol.2007; 203:63-71.
27 Dong J, Yin H, Liu W, et al. Congenital iodine deficiency and hypothyroidism impair LTP and decrease c-fos and c-jun expression in rat hippocampus. Neurotoxicology.2005; 26: 417-426.
28 Gerges NZ, Alkadhi KA. Hypothyroidism impairs late LTP in CA1 region but not in dentate gyrus of the intact rat hippocampus:MAPK involvement. Hippocampus.2004; 14:40-45.
29 Gilbert ME, Sui L, Walker MJ, et al. Thyroid hormone insufficiency during brain development reduces parvalbumin immunoreactivity and inhibitory function in the hippocampus. Endocrinology.2007; 148:92-102.
30 Sui L, Anderson WL, Gilbert ME. Impairment in short-term but enhanced long-term synaptic potentiation and ERK activation in adult hippocampus area CA1 following developmental thyroid hormone insufficiency. Toxicol Sci.2005; 85:647-656.
31 Navarro A, Anand-Apte B, Parat MO. A role for caveolae in cell migration. FASEB J.2004; 18:1801-1811.
32 von Bohlen Und Halbach O. Immunohistological markers for staging neurogenesis in adult hippocampus. Cell Tissue Res.2007; 329(3):409-420.
33 Gleeson JG, Allen KM, Fox JW, et al. Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein. Cell. 1998; 92(1):63-72.
34 Koizumi H, Higginbotham H, Poon T, et al. Doublecortin maintains bipolar shape and nuclear translocation during migration in the adult forebrain. Nat Neurosci.2006; 9(6): 779-786.
35 Koshibu K, Levitt P, Ahrens ET. Sex-specific, postpuberty changes in mouse brain structures revealed by three-dimensional magnetic resonance microscopy. Neuroimage.2004; 22(4): 1636-1645.
36 Lenroot RK, Giedd JN. Brain development in children and adolescents:insights from anatomical magnetic resonance imaging. Neurosci Biobehav Rev.2006; 30(6):718-729.
37 Aimone JB, Wiles J, Gage FH. Potential role for adult neurogenesis in the encoding of time in new memories. Nat Neurosci.2006; 9(6):723-727.
38 Farioli-Vecchioli S, Saraulli D, Costanzi M, et al. The timing of differentiation of adult hippocampal neurons is crucial for spatial memory. PLoS Biol.2008; 6(10):e246.
39 Prickaerts J, Koopmans G, Blockland A, et al. Learning and adult neurogenesis:survival with or without proliferation? Neurobiol Learn Mem.2004; 81:1-11.
40 Sokolov EN, Nezlina NI. Long-term memory, neurogenesis, and signal novelty. Neurosci Behav Physiol.2004; 34:847-857.
41 Sala-Roca J, Estebanez-Perpina E, Balada F, et al. Effects of adult dysthyroidism on the morphology of hippocampal neurons. Behav Brain Res.2008; 188(2):348-354.
42 Prickaerts J, Koopmans G, Blokland A, et al. Learning and adult neurogenesis:survival with or without proliferation? Neurobiol Learn Mem.2004; 81(1):1-11.
43 Gould E, Tanapat P, Hastings NB, et al. Neurogenesis in adulthood:a possible role in learning. Trends Cogn Sci.1999; 3(5):186-192.
44 Joo JY, Kim BW, Lee JS, et al. Activation of NMDA receptors increases proliferation and differentiation of hippocampal neural progenitor cells. J Cell Sci.2007; 120(Pt 8):1358-1370.
45 Markakis EA, Gage FH. Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J Comp Neurol.1999; 406(4):449-460.
46 van Praag H, Schinder AF, Christie BR, et al. Functional neurogenesis in the adult hippocampus. Nature.2002; 415(6875):1030-1034.
47 Kempermann G, Wiskott L, Gage FH. Functional significance of adult neurogenesis. Curr Opin Neurobiol.2004; 14(2):186-191.
48 Kesner RP, Rogers J. An analysis of independence and interactions of brain substrates that subserve multiple attributes, memory systems, and underlying processes. Neurobiol Learn Mem. 2004; 82(3):199-215.
49 Treves A, Rolls ET. Computational analysis of the role of the hippocampus in memory. Hippocampus.1994; 4(3):374-391.
50 Leuner B, Waddell J, Gould E, et al. Temporal discontiguity is neither necessary nor sufficient for learning-induced effects on adult neurogenesis. J Neurosci.2006; 26(52):13437-13442.
51 Takeda A, Yamada K, Tamano H, et al. Hippocampal calcium dyshomeostasis and long-term potentiation in 2-week zinc deficiency. Neurochem Int.2008; 52:241-246.
52 Wang FD, Bian W, Kong LW, et al. maternal zinc deficiency impairs brain nestin expression in prenatal and postnatal mice. Cell Res.2001; 11:135-141.
53 Gdalyahu A, Ghosh I, Levy T, et al. DCX, a new mediator of the JNK pathway. EMBO J. 2004; 23(4):823-832.
54 Moores CA, Perderiset M, Kappeler C, et al. Distinct roles of doublecortin modulating the microtubule cytoskeleton. EMBO J.2006; 25(19):4448-4457.
55 Royland JE, Parker JS, Gilbert ME. A genomic analysis of subclinical hypothyroidism in hippocampus and neocortex of the developing rat brain. J Neuroendocrinol.2008; 20(12): 1319-1338.
56 Doberty P, Walsh FS. CAM-FGF receptor interations:a model for axonal growth. Mol Cell Neurosci.1996; 8(1):99-111.
57 Schuster T, Krug M, Hassan H, et al. Increase in proportion of hippocampal spine synapses expressing neural cell adhesion molecule NCAM180 following long-term potentiation. J Neurobiol.1998; 37(3):359-372.
58 Kasper C, Rasmusen H, Kastrup JS, et al. Structural basis of cell-cell adhesion by NCAM. Nature Structural Biol.2000; 7:387-393.
59 Eckhardt M, Bukalo O, Chazal G, et al. Mice deficient in the polysialyltransferase ST8SiaⅣ/PST-1 allow discrimination of the roles of neural cell adhesion molecule protein and polysialic acid in neural development and synaptic plasticity. J Neurosci.2000; 20(14): 5234-5244.
60 Walmod PS, Kolkovak, Berezin V, et al. Zippers make signals NCAM-mediated molecular interactions and signal transduction. Neurochem Res.2004; 29(11):2015-2035.
61 Auso E, Lavado-Autric R, Cuevas E, et al. A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration. Endocrinology.2004; 145(9):4037-4047.
62 Farwell AP, Dubord-Tomasetti SA. Thyroid hormone regulates the expression of laminin in the developing rat cerebellum. Endocrinology.1999; 140(9):4221-4227.
63 Ronn LC, Berezin V, Bock E. The neural cell adhesion molecule in synaptic plasticity and ageing. Int J Dev Neurosci.2000; 18(2-3):193-199.
64 Iglesias T, Caubin J, Stunnenberg HG, et al. Thyroid hormone-dependent transcriptional repression of neural cell adhesion molecule during brain maturation. EMBO J.1996; 15(16): 4307-4316.
65 Tarsa L, Goda Y. Synaptophysin regulates activity-dependent synapse formation in cultured hippocampal neurons. PNAS.2002; 99(2):1012-1016.
66 Gomez M, Hernandez ML, Pazos MR, et al. Colocalization of CB1 receptors with L1 and GAP-43 in forebrain white matter regions during fetal rat brain development:evidence for a role of these receptors in axonal growth and guidance. Neuroscience.2008; 153:687-699.
67 Dzienis-Koronkiewicz E, Debek W, Chyczewski L. Use of synaptophysin immunohistochemistry in intestinal motility disorders. Eur J Pediatr Surg.2005; 15 (6): 392-398.
68 Scarfone E, Dememes D, Sans A. Synapsin I and Synaptophysin expression during ontogenesis of the mouse peripheral vestibular system. J Neurosci.1991; 11:1173-1181.
69 Liebau S, Vaida B, Storch A, et al. Maturation of synaptic contacts in differentiating neural stem cells. Stem Cells.2007; 25:1720-1729.
70 Ferreira A, Kosik KS, Greengard P, et al. Aberrant neurites and synaptic vesicle protein deficiency in synapsin Ⅱ-depleted neurons. Science.1994; 264(5161):977-979.
71 Chin LS, Li L, Ferreira A, et al. Impairment of axonao development and of aynaptogenesis in hippocampal neurons of synapsin I-deficient mice. Pro Natl Acad Sci USA.1995; 92: 9230-9234
72 Angers A, Fioravante D, Chin J, et al. Serotonin stimulates phosphorylation of aplysia synapsin and alters its subcellular distribution in sensory neurons. J Neurosci.2002; 22(13): 5412-5422.
73 Fiala JC, Feinberg M, Popov V, et al. Synaptogenesis via dendritic filopodia in developing hippocampal area CA1. J Neurosci.1998; 18:8900-8911.
74 Nikizad H, Yon JH, Carter LB, et al. Early exposure to general anesthesia causes significant neuronal deletion in the developing rat brain. Ann N Y Acad Sci.2007; 1122:69-82.
75 Bernal J. Action of thyroid hormone in brain. J Endocrinol Invest.2002; 25:268-288.
76 Dong H, Yauk CL, Rowan-Carroll A, et al. Identification of thyroid hormone receptor binding sites and target genes using ChIP-on-chip in developing mouse cerebellum. PLoS One.2009; 4(2):e4610.