大鼠视皮层中脑源性神经营养因子及γ-氨基丁酸表达的年龄相关性变化

目的 比较不同年龄组大鼠初级视皮层中脑源性神经营养因子（BDNF）及抑制性神经递质γ-氨基丁酸（GABA）表达的年龄相关性变化，为探讨老年性视觉功能衰退的细胞分子机制提供线索。方法 Nissl染色显示视皮层分层并用于神经元计数；免疫组织化学法标记大鼠视皮层中BDNF及GABA免疫阳性神经元。光镜下观察并用Image-Pro Express 6.0分析软件进行细胞密度统计和免疫反应吸光度值测量。结果 青年、中年及老年组(每组n=6)大鼠初级视皮层各层的神经元平均密度差异不显著；但与青年组大鼠相比，中年组及老年组大鼠初级视皮层各层中BDNF及GABA免疫阳性神经元密度显著下降，BDNF及GABA免疫反应强度明显减弱；与中年组大鼠相比，老年组大鼠视皮层各层中BDNF及GABA免疫阳性神经元密度及其反应强度亦明显降低。在衰老过程中，大鼠初级视皮层各层内GABA表达的减少与BDNF表达的降低具有高度的相关性。结论 大鼠初级视皮层中BDNF和GABA的表达随着衰老出现进行性下降，衰老过程中BDNF分泌的减少可能引起脑内抑制性神经递质GABA表达下调，这可能是介导老年性视觉功能衰退的重要途径之一。     

猫初级听皮层γ-氨基丁酸能神经元和星形胶质细胞年龄相关变化

目的探讨青年猫和老年猫初级听皮层（AI）的年龄相关变化及可能产生的生理作用。方法Nissl染色示两组猫初级听皮层的分层结构并进行细胞计数;免疫组织化学染色示两组猫7．氨基丁酸（GABA）能神经元和星形胶质细胞的形态、密度及分布。结果与青年猫相比,老年猫的初级听皮层细胞平均密度无显著的年龄相关变化,GABA-IR细胞的密度显著下降（尤其是Ⅱ、Ⅲ、Ⅳ层）,阳性胞体较小,阳性反应明显减弱;老年猫的GFAP-IR细胞密度显著增大,阳性胞体膨大,突起稠密粗大,阳性反应明显增强,其中的Ⅰ层和白质尤为明显。结论GABA能神经元显著的年龄相关性改变可能为老年性听觉功能衰退的神经机制在皮层水平提供了直接的形态学证据;而星形胶质细胞活动增强可能是导致GABA能神经元及GABA递质含量减少的重要原因之一。     

猫初级视区急性毁损对高级视区刺激诱导的c-fos蛋白表达的影响

目的:研究初级视区急性毁损对猫的高级视区刺激诱导的c-fos蛋白表达的影响,探索初级视区毁损后的残留视觉(称盲视)可能机制。方法:本研究采用免疫组织化学检测了初级视区(包括17和18区)急性毁损对猫的高级视区(包括19、20和21区)中立早基因c-fos蛋白基础表达量和刺激诱导的c-fos蛋白表达量的影响,以正常猫为对照。结果:急性毁损初级视区后,高级视区内c-fos免疫阳性神经元密度及c-fos免疫阳性反应的光吸收值(OA)在初级视区毁损猫和正常对照猫之间无显著差异;然而,双侧初级视区毁损猫的高级视区中刺激诱导的c-fos免疫阳性神经元密度比正常猫下降了92%以上,c-fos蛋白免疫阳性反应的OA值下降了78.9%以上。结论:初级视区急性毁损后高级视区的绝大多数神经元失去了对视觉刺激的反应性,因此,初级视区毁损后出现的盲视现象可能主要与皮层下中枢至高级视区的神经通路重组有关。     

猫年龄相关的视网膜γ-氨基丁酸和神经丝蛋白表达

目的：对老年猫与青年猫视网膜的变化进行比较研究。方法：运用免疫组织化学ABC法显示卜氨基丁酸（GABA）、神经丝蛋白（NF）阳性结构。显微镜下观察GABA、NF阳性反应,并计阳性细胞数。结果：老年猫、青年猫GABA免疫反应阳性结构均见于无长突细胞、内网状层、神经节细胞和神经纤维层。老年猫NF阳性结构见于水平细胞、无长突细胞以及神经节细胞。青年猫无长突细胞未见阳性反应。与青年猫相比较,老年猫视网膜中GABA、NF阳性反应强于青年猫,免疫反应阳性细胞显著增加。结论：老年猫视网膜在衰老过程中呈现年龄相关的结构改变,这可能是造成视觉功能衰退的重要因素之一。     

P>猫皮层第一体感区髓质胶质反应的年龄相关性变化/P>

目的 比较青年猫与老年猫大脑皮层第一躯体感觉区(SI区)髓质中胶质细胞和S100蛋白免疫阳性细胞和胶质纤维酸性蛋白(GFAP)免疫阳性细胞的年龄相关性形态学变化, 探讨该变化的原因及其在老年性躯体感觉功能衰退中的意义.方法 选青年猫与老年猫各4只,用改良的Holzer结晶紫染色显示SI区髓质中所有胶质细胞,并用成年动物Golgi法显示其形态;免疫组织化学法(SABC法)显示S100蛋白免疫阳性(S100-IR)细胞及GFAP免疫阳性(GFAP-IR)细胞.光镜下观察细胞形态,用BI-2000医学图像分析系统计数SI区髓质中的胶质细胞、S100-IR细胞及GFAP-IR细胞的数量,测定S100及GFAP表达的平均吸光度值和阳性细胞面积比.结果 与青年猫相比,老年猫SI区髓质中胶质细胞、S100-IR细胞及GFAP-IR细胞密度显著增加(P<0.01),S100-IR细胞及GFAP-IR细胞占胶质细胞的比例均增加,免疫组织化学染色平均吸光度值及阳性细胞面积比也显著增大(P<0.01);星形胶质细胞胞体膨大,突起稠密、粗大.结论 衰老过程中,猫SI区髓质中存在明显的反应性胶质化, 其中星形胶质细胞对衰老更为敏感.     

NGF、BDNF及受体trkA、trkB、trkC在正常猴脊髓的表达

采用免疫组织化学方法观察了神经生长因子 (NGF) ,脑源性神经营养因子 (BDNF)以及 NGF家族因子受体 trk A、trk B、trk C的免疫阳性反应在正常猴脊髓的分布。结果表明 :NGF免疫反应阳性的神经元在脊髓灰质各层中均有分布 ,灰、白质内也可见较多的 NGF免疫反应阳性的胶质细胞。 BDNF在脊髓各型神经无有明显的表达 ,特别是前角运动神经元。 trk A、trk B、trk C的免疫阳性反应产物主要分布在灰质的神经元及胶质细胞。本实验结果揭示了在正常猴脊髓中神经营养因子 (NGF、BDNF )及受体 trk A、trk B、trk C的表达状况 ,提示这些神经营养因子及受体在维持猴脊髓神经元的正常生理功能中具有重要作用。     

猫小脑皮质结构的年龄相关性变化

目的：对青年和老年猫小脑皮质结构的年龄性变化进行比较。方法：动用Nissl染色显示小脑皮质神经元,免疫组织化学方法显示胶质纤维酸性蛋白免疫阳性（GFAP-IR）星形胶质细胞和神经丝蛋白免疫阳性（NF-IR）结构。显微镜下观察测量小脑皮质厚度和细胞密度。结果：与青年猫比较,老年猫小脑皮质总厚度及分子层厚度显著下降,颗粒层厚度明显增加,各层神经元密度明显降低;颗粒层中GFAP-IR细胞密度显著增加,阳性反应增强;老年猫蒲肯野细胞（PC）NF免疫阳性树突分支明显减少。结论：衰老过程中小脑皮质神经元丢失和PC中NF阳性树突减少,可能会导致老年小脑皮质接受和整合信息的功能降低,而星形胶质细胞活动增强对皮质神经元可能起保护作用。     

硫酸软骨素蛋白多糖对视皮层γ-氨基丁酸能神经元表达及其抑制性突触传递功能的影响

目的:通过体外观察硫酸软骨素蛋白多糖(CSPGs)对γ-氨基丁酸(GABA)能神经元表达及其抑制性突触传递功能的影响,为探索CSPGs抑制视皮层可塑性的机制提供实验依据。方法:运用硫酸软骨素酶(ChABC)处理体外培养的胎鼠视皮层神经元,降解其CSPGs后应用免疫荧光显色检测CSPGs降解情况及GABA能神经元的表达变化,并用膜片钳检测其自发性微小性抑制性突触后电流(mIPSCs)以观察其抑制性突触传递功能变化情况。结果:0.1 U/ml ChABC处理神经元后,CSPGs被成功降解,GABA能神经元细胞密度、mIPSCs幅度和频率均显著低于正常对照组。结论:CSPGs能促进GABA能神经元表达及其抑制性突触传递功能的成熟,这可能是CSPGs抑制视皮层可塑性的作用机制之一。     

BDNF对体外培养的大鼠脊髓前角神经元内突触素Ⅰ与突触囊泡素表达的影响

探讨BDNF对体外培养的大鼠脊髓前角神经元内突触素I与突触囊泡素(SYN)表达的影响。取孕14d大鼠子宫内胎鼠的脊髓腹侧部分神经元,体外有血清培养。在培养7d后,随机分成对照组、BDNF组和抗BDNF组。BDNF组培养液中加入BDNF(20ng/ml),抗BDNF组培养液中加入BDNF抗体(20μg/ml),对照组加入等量Hanks液。3d后在倒置显微镜下计数三组神经元存活数,并用NF200、MAP2、NSE的免疫组化反应对神经细胞进行鉴定。行突触素I与SYN免疫组化反应,对部分细胞行突触素ImRNA原位杂交反应,运用图像分析系统对突触素I与SYN免疫组织反应阳性产物以及突触素I原位杂交反应阳性产物作光密度分析。结果发现有血清培养时各组脊髓前角神经元的存活数无显著差异(P>0.05);BDNF组突触素I与SYN免疫反应阳性产物的平均光密度值高于其它两组,抗BDNF组最低(P<0.01)。BDNF组突触素ImRNA阳性产物的平均光密度值明显高于其它两组,抗BDNF组突触素ImRNA阳性产物的平均光密度值最低(P<0.01)。本研究结果提示BDNF对有血清培养时脊髓前角神经元的存活没有明显影响,但BDNF可明显上调培养的脊髓前角神经元内突触素I与SYN的表达。     

脑源性神经营养因子在早期人胚神经管发育中的定位表达

目的研究脑源性神经营养因子(BDNF)在早期人胚神经管发育过程中的定位表达。方法采用免疫细胞化学ABC法染色,研究35天人胚的发育情况。结果在人胚神经管的室带中,神经元的细胞质BDNF免疫反应阳性;在中间带,神经元的突起BDNF免疫反应阳性,一部分神经元的细胞核BDNF免疫反应阳性,另外一部分神经元的细胞核BDNF免疫反应阴性;在缘带BDNF的分布与中间带相似。神经管的头侧较尾侧BDNF阳性反应较强,神经管的腹侧BDNF阳性反应较背侧强。结论BDNF在人胚神经管免疫反应阳性,表明BDNF是诱导神经管分化发育的重要信号分子,提示BDNF在人胚神经管的发育中具有十分重要的作用。     

Quantitative distribution of GABA-immunoreactive neurons in the visual cortex (area 17) of the cat

Summary Cortical neurons using the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) are known to contribute to the formation of neuronal receptive field properties in the primary visual cortex (area 17) of the cat. In order to determine the cortical location of GABA containing neurons and what proportion of cortical neurons might use GABA as their transmitter, we analysed their distribution quantitatively using a post-embedding GABA immunohistochemical method on semithin sections in conjunction with stereological procedures. The mean total numerical density of neurons in the medial bank of the lateral gyrus (area 17) of five adult cats was 54,210±634 per mm³ (¯x±SD). An average of 20.60±0.48% (¯x±SEM) of the neurons were immunoreactive for GABA. The density of GABA-immunoreactive neurons was somewhat higher in layers II, III and upper VI, compared with layers I, IV, V and lower VI, with the lowest density being in layer V. The proportion of GABA-immunopositive cells relative to immunonegative neurons gradually decreased from the pia to the white matter. Layer I was different from other layers in that approximately 95% of its neurons were GABA-immunoreactive. The results allowed the calculation of the absolute numbers of GABAergic neurons in each layer under a given cortical surface area and could provide the basis for the quantitative treatment of cortical circuits.     

Changes of μ-opioid receptors and GABA in visual cortex of chronic morphine treated rats

Electrophysiological and biochemical studies have indicated that GABAergic modulation is involved in the opioid-induced altering of response properties of visual cortical cells and impairing of short-term synaptic plasticity in the geniculo-cortical visual pathway. The aim of the current study was to examine whether there were changes in the localization and density of μ-opioid receptor subtype (MOR1) and GABA in the visual cortex of morphine-dependent and abstinent rats. Immunofluorescence histochemical method was applied to display MOR1 and GABA distribution. We found that MOR1-like immunoreactive neurons were significantly lowered in layer I–VI of visual cortex of rats sacrificed immediately after the last injection (defined as morphine-dependent (MD)) than saline-control group. In rats sacrificed just before the last injection (defined as morphine-abstinent (MA)), the density of μ-opioid receptor was higher than that in dependent group in layer I–V neurons of visual cortex, but remained lower than those in control group. Three hours after the last morphine injection (defined as 3 h after morphine-abstinent (3 h)), MOR1-like immunoreactive neurons in layer I and layer IV of visual cortex were still significantly lower than control. As to GABA-like immunoreactive neurons, they were significantly decreased in abstinent group compared to dependent group. These results provide morphological evidence that opioid-induced altering of response properties of visual cortical cells might be due to the change of visual cortical GABAergic system induced by opioid via μ-opioid receptor, which result in disinhibition of visual cortex projection neurons and their abnormal firing.     

Impaired GABAergic inhibition in the visual cortex of brain-derived neurotrophic factor heterozygous knockout mice

Brain derived neurotrophic factor (BDNF) promotes the formation, maturation and stabilization of inhibitory synapses in the central nervous system. In addition, BDNF has been suggested to regulate the critical period for ocular dominance plasticity in the visual system. Here we further evaluated the role of BDNF in the visual cortex by studying the GABAergic synaptic transmission under conditions of chronically reduced levels of BDNF. Whole-cell patch-clamp recordings were performed from pyramidal neurons located in layers II/III of visual cortical slices in heterozygous BDNF knockout mice (BDNF (+/-)) and their wild-type littermates at the age of 21-25 days. The BDNF (+/-) mice showed a decreased frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) as well as a reduced amplitude and prolonged decay time constant of evoked IPSCs. Further analyses indicated an impaired presynaptic GABAergic function in BDNF (+/-) mice, as shown by the decreased release probability, steady-state release and synchronous release of GABA. However, the number of functional release sites remained unchanged. In line with these observations, an impaired glutamate-driven GABA release was observed in BDNF (+/-) mice. Furthermore, the overall balance in the strength of cortical excitation to inhibition shifted towards a decreased inhibition. Finally, the reversal potential for chloride-mediated evoked IPSCs was not affected. These findings suggested that chronically reduced levels of BDNF strongly impair the GABAergic inhibitory function in visual cortex by altering postsynaptic properties and by reducing presynaptic GABA release as well as the overall strength of inhibition onto pyramidal neurons within the cortical network. These impairments of inhibitory function are compatible with a rather immature status of the GABAergic system in BDNF (+/-) mice, which supports the hypothesis that the level of expression for BDNF critically affects maturation and function of the GABAergic inhibition.     

Degradation of response modulation of visual cortical cells in cats with chronic exposure to morphine

The primary visual cortex (V1) plays an important role in vision and visual perception. Studies in many brain regions demonstrate that opiate abuse can change excitatory and inhibitory neurotransmission. To investigate the effect of chronic morphine exposure on the response modulation of V1 simple and complex neurons, we carried out in vivo extracellular recordings in V1 of morphine- and saline-treated (control) cats. Response modulation was quantified as the ratio of first Fourier components (F1) to the mean response (F0). Compared with saline-treated cats, V1 neurons in morphine-treated cats exhibited weaker response modulation and a longer time course of response. The decrease of response modulation was caused by an increase of F0. Further, morphine re-exposure significantly improved the response properties of V1 neurons in morphine-treated cats. These results suggest that chronic morphine treatment leads to a significant degradation of response modulation of V1 neurons and a morphine dependence of primary visual cortical function.     

葛根素对血管性痴呆大鼠海马锥体细胞和BDNF表达的影响

为了观察葛根素对血管性痴呆(VD)模型大鼠海马锥体细胞和BDNF表达的影响及其作用机制,本研究采用双侧颈总动脉缺血再灌注,同时腹腔注射硝普钠建立血管性痴呆大鼠模型,选出造模成功者随机分为模型组及葛根素干预组,各为24只,另以条件匹配的24只大鼠为假手术组。分别在造模术后15d,1、2和4个月等时间点,采用水迷宫检测大鼠学习记忆能力的变化,HE染色和免疫组化染色观察大鼠海马神经元的形态学改变及BDNF表达的变化。结果显示:(1)模型组大鼠的逃逸潜伏期(EL)均明显长于假手术组(P<0.01),葛根素干预组大鼠的EL较模型组明显缩短(P<0.05),但仍长于假手术组(P<0.05);(2)模型组大鼠海马CA1区锥体细胞数比假手术组明显减少(P<0.01),葛根素干预组2个月和4个月时点锥体细胞数较模型组明显增多(P<0.01),但仍少于假手术组(P<0.01);(3)模型组大鼠海马BDNF阳性细胞明显减少(P<0.01),除15d和1个月组DG区外,葛根素干预组大鼠海马BDNF阳性细胞数较模型组明显增多(P<0.05),但仍低于假手术组(P<0.05);(4)模型组大鼠海马BDNF阳性细胞平均吸光度值较假手术组明显降低(P<0.01),而葛根素干预组比模型组和假手术组均明显降低(P<0.05)。本研究结果提示,脑缺血再灌注后,海马BDNF阳性神经元和锥体细胞持续减少,在VD学习记忆障碍的发生和发展过程中起重要作用;葛根素对脑缺血再灌注损伤具有保护作用,其机制可能与葛根素上调BDNF的表达、减少锥体细胞的丢失有关。     

Investigation of the morphometric parameters of the visual cortical region (Field 17) in perinatal brain damage

Autopsy brain specimens from babies who had experienced perinatal central nervous system damage and those who had shown no symptoms of brain lesion (a control group) were used to study the structural characteristics of Field 17 of the visual cortical region, by using computed morphometry. As compared with the control group, the children who had suffered from perinatal pathology showed no relationship between the increased width of a cortical plate diameter and the baby's age and Layers I and V had a significantly less width. In most cases Layers III, V and Layer VI + VII contained smaller neurons and the density of their arrangement was much less than in the brain of the control babies. It was concluded that the detected disorders suggest growth retardation and nonspecific structural and functional changes in the visual cortex in perinatal brain damage.     

Development of dendritic bundles of pyramidal neurons in the rat visual cortex.

The apical dendrites of pyramidal neurons in the cerebral cortex form vertical bundles whose distribution and density vary across species and areas. To understand their relationships with cortical columns, we labeled retrogradely neurons from the white matter underlying the visual cortex with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) at P3 and P10 and with biotinylated dextran amine at P30. We also mapped the distribution of apical dendrites in tangential sections, immunostained for microtubule-associated proteins (MAP2). Their composition and distribution were studied with Neurolucida and NeuroExplorer software. The apical dendrites of pyramidal neurons formed different bundle types: at P3 we found bundles formed (a) by neurons located in cortical plate; (b) by layer V neurons; and (c) by upper layer V neurons and cortical plate neurons. At P10, the amount of supragranular neurons participating in the bundles increased. The inter-dendritic and inter-bundle distances increased with age. These findings confirm that dendritic bundles are present in the rat visual cortex early in development and are formed by neurons belonging to different cortical layers. The existence of different types of bundles relative to the layer of location of their parent neurons suggests that they are heterogeneous from each other in nature and in the pattern of connectivity.     

The effect of repetitive spreading depression on neuronal damage in juvenile rat brain

Spreading depression (SD) is pronounced depolarization of neurons and glia that travels slowly across brain tissue followed by massive redistribution of ions between intra- and extracellular compartments. There is a relationship between SD and some neurological disorders. In the present study the effects of repetitive SD on neuronal damage in cortical and subcortical regions of juvenile rat brain were investigated. The animals were anesthetized and the electrodes as well as cannula were implanted over the brain. SD-like event was induced by KCl injection. The brains were removed after 2 or 4 weeks after induction of 2 or 4 SD-like waves (with interval of 1 week), respectively. Normal saline was injected instead of KCl in sham group. For stereological study, paraffin-embedded brains were cut in 5 μm sections. The sections were stained with Toluidine Blue to measure the volume-weighted mean volume of normal neurons and the numerical density of dark neurons. The volume-weighted mean volume of normal neurons in the granular layer of the dentate gyrus and layer V of the temporal cortex in SD group were significantly decreased after four repetitive SD. Furthermore, densities of dark neurons in the granular layer of the dentate gyrus (after 2 weeks), the caudate–putamen, and layer V of the temporal cortex (after 4 weeks) were significantly increased in SD group. Repetitive cortical SD in juvenile rats may cause neuronal damage in cortical and subcortical areas of the brain. This may important in pathophysiology of SD-related neurological disorders.     

Birth-date dependent alignment of GABAergic neurons occurs in a different pattern from that of non-GABAergic neurons in the developing mouse visual cortex

In the developing mouse cerebral cortex, gamma-aminobutyric acid (GABA)ergic neurons and non-GABAergic neurons arise in distinct places and migrate into the cortical plate (CP) via different pathways. Although the "inside-out" alignment of projection neurons in the cortex has been thoroughly analyzed, the pattern of interneuron alignment is not well understood. Herein, we show that in the postnatal day (P) 9.5 mouse visual cortex, GABAergic neurons born on embryonic day (E) 12.5 were distributed around two peak locations, mainly around layer V and also around layer II/III, while non-GABAergic neurons born on E12.5 were distributed around only one peak in layer VI. Both cell populations born on E15.5 exhibited only one common peak distribution in layer II/III. The two peak locations of GABAergic neurons born on E12.5 still existed at P30. When the subtypes of GABAergic neurons were analyzed, calretinin-positive cells born on E12.5 were distributed in the cortex around one peak location near layer II/III, whereas somatostatin-positive E12.5 cells were distributed in the cortex around one peak location near layer V. These results suggest that the alignment of interneurons is regulated differently according to subtypes and from that of projection neurons having the same embryonic day of origin.