SD大鼠视网膜切片的视杆双极细胞L-钙通道电流的研究

目的 研究SD大鼠视网膜双极细胞(RBCs)L-钙通道电流的特点.方法 选取11只4～8周龄SD大鼠制备视网膜切片标本,RBCs胞体位于视网膜内核层最外部,以辨别细胞.使用全细胞膜片钳的方法,记录视网膜RBCs的L-钙通道电流.结果 在显微镜下从切片上识别位于视网膜内核层最外部的RBCs胞体.用玻璃毛细管微电极在RBCs 胞体记录出L-钙通道电流,慢电容补偿:(3.2±0.3)pF,阶越(step)刺激的电流峰值:(31.1±3.3)pA,斜坡(ramp)刺激的电流峰值:(32.5±3.1)pA;使用L-钙通道阻断剂nifedipine可以成功阻断电流.结论 实验中记录出的电流为L-钙通道电流.使用全细胞膜片钳的方法在大鼠视网膜切片上记录RBCs的L-钙通道电流是可行的.     

慢性眼内压增高对大鼠视网膜Muller细胞钙通道电流的影响

目的利用电生理方法,鉴定大鼠视网膜Muller细胞钙通道,进而研究慢性眼内压增高对Muller细胞钙通道电流的影响。方法在急性分离的大鼠Muller细胞上,利用全细胞膜片钳的电压钳技术记录钙通道电流。采用结扎巩膜上静脉的方法制备大鼠高眼压模型。结果当细胞外液中不含二价阳离子时,可以记录到电流幅度较大的钙通道介导的Na＋流。该电流可被L-型钙通道阻断剂nimodipine和T-钙通道阻断剂mibefradil可逆地压抑到加药前的（39．8±5．4）％（P〈0．001）和（46．7±8．7）％（P〈0．001）。与假手术组相比,高眼压术后1周、2周和4周的大鼠视网膜Mtiller细胞钙通道的电流幅度没有明显的变化。然而,电流成分分析发现,与对照组相比,高眼压大鼠Muller细胞的nimodipine敏感电流呈降低的趋势[1周：（70,9±13．3）％;2周：（70．5±21．9）％;4周：（69．2±23．9）％],而mibefradil敏感的电流在高眼压术后1周和2周呈增高趋势[（157．5±21．2）％和（158．6±35．5）％],4周时趋于正常（109．2±37．9）％。结论大鼠视网膜Mailer细胞功能性表达L-型和T-型钙通道。慢性眼内压增高导致L-型钙通道电流减小,T-型钙通道电流增大,从而增加胞内钙,共同参与Mtiller细胞的去极化和激活。     

改良法制作的视网膜切片内核层神经元的形态和电学特性

目的:探讨低温琼脂包埋振动切片机切片法制作的大鼠视网膜切片内核层神经元的形态和基本电生理学特性。方法:采用低温琼脂包埋振动切片机切片的方法制作大鼠视网膜切片,对内核层的神经元进行膜片钳全细胞记录,同时在胞内液中加入荧光黄观察记录细胞的形态。结果:该方法制作的视网膜切片切面平整、细胞活性好、保留了细胞之间的突起联系,能够根据细胞胞体的大小、位置初步辨别细胞的种类。在视网膜切片上荧光黄显示的细胞形态表明,双极细胞胞体呈梭形,突起主要沿纵向延伸;而水平细胞和无长突细胞胞体圆形或椭圆形、胞体较大,分别位于内核层的最外层和最内层。水平细胞和无长突细胞的静息膜电位(RMP)和膜电容(Cm)明显高于双极细胞。给予时程40ms,步阶10mV从-60mV至+40mV的电压刺激,41.7%的视锥双极细胞和64.7%的无长突细胞表现出内向钠电流和外向钾电流,其他细胞则只表现出外向钾电流。结论:采用低温琼脂包埋振动切片机切片的方法操作简单,制作的切片质量稳定可靠,使得在视网膜切片上对包括水平细胞在内的不同内核层神经元进行膜片钳记录成为可能。进一步研究视网膜内核层神经元的电生理学特性,有助于揭示视觉信号的发生、传导和调控机制。     

人和小鼠视网膜双极细胞的形态及功能比较

目的比较人和小鼠视网膜视杆双极细胞形态和药物模拟对光反应的电流特性。方法实验研究。对视网膜冰冻切片行免疫荧光染色,用PKC-α抗体标记视网膜视杆双极细胞,以观察其形态分布特点。在灌流充氧的情况下制备视网膜薄片切片,行视网膜ON型以及OFF型双极细胞的全细胞膜片钳记录。分别在ON型双极细胞和OFF型双极细胞上给予快速加药40 µmol/L LY3414925或100 µmol/L AMPA,诱导出谷氨酸电流,以记录双极细胞模拟对光反应,并比较人和小鼠的谷氨酸电流动力学的差异,人和小鼠各项数据比较采用非参数检验（Mann-Whitney检验）进行处理。结果人和小鼠的视网膜视杆双极细胞形态分布相似,但PKC-α表达略有不同。膜片钳结果显示人视网膜ON型双极细胞的模拟对光反应的上升相的达峰时间为（1.91±0.11）ms,与小鼠视网膜ON型双极细胞[（0.83±0.08）ms]相比明显较长（U=0.00,P＜0.01）,而人视网膜ON型双极细胞的模拟对光反应恢复时间为（1.34±0.40）ms,明显短于小鼠[（20.06±3.07）ms]（U=0.00,P＜0.01）。但人和小鼠视网膜OFF型双极细胞电流动力学即电流幅度[人：（143.0±2.1）pA;小鼠：（136.3±2.2）pA]、反应上升时间[人：（1.91±0.35）ms;小鼠：（1.28±0.52）ms]和恢复时间[人：（220.5±10.8）ms;小鼠：（168.1±29.3）ms]差异均无统计学意义。结论 视杆双极细胞在人和小鼠视网膜中分布位置和形态大致相似。人和小鼠视网膜ON型双极细胞电流特性有差异。     

遗传性视网膜病变模式大鼠的氧诱导视网膜病变观察

目的:探索遗传性视网膜病变模式大鼠的氧诱导视网膜新生血管特点。方法:将新生(生后7d)的SD大鼠、CSNB大鼠和RCD大鼠各1窝每窝(即每组)6只暴露于800±20mL/L氧浓度环境中持续饲养5d,然后再回到正常氧环境条件饲养5d;对照组为以上3种品系同龄新生鼠各1窝,每窝(即每组)各6只置于正常氧环境中饲养17d作为对照。在第18d时将所有幼鼠行心脏墨汁灌注,取出两侧眼球,其中1眼用于视网膜铺片了解视网膜血管形态的改变,另1眼用于组织切片观察并统计突破视网膜内界膜的血管内皮细胞核的数目。结果:在正常饲养环境下,RCD大鼠视网膜血管网稀疏,SD大鼠CSNB大鼠视网膜血管未见明显异常。在氧诱导下,各实验组大鼠视网膜血管正常网络状结构受到破坏,结构稀疏,血管迂曲、收缩或扩张,部分血管出现闭塞,其中SD大鼠、CSNB大鼠视网膜有出血点,甚至片状出血。组织切片显示对照组的SD大鼠中偶见突破内界膜的内皮细胞核,其他两组均未见。实验组中SD大鼠、CSNB大鼠和RCD大鼠3种品系中均可见较多的突破内界膜内皮细胞核,计数结果分别为24.10±2.49,38.20±10.47,68.00±3.06,与同品系的对照组有显著性的差异(P<0.01);与不同品系的实验组比较也均有显著性差异(P<0.01)。结论:氧诱导的新生大鼠视网膜新生血管与品系有关,视网膜退行性病变大鼠仍可诱导出视网膜新生血管,且严重程度可能与感光细胞的功能有关。     

小鼠视觉发育前关键期视皮层神经元反应特性与突触可塑性

背景 人类及哺乳动物的视觉发育主要是在生后关键期内完成的,但此期并非是哺乳动物接受视觉经验刺激的最早期.小鼠等哺乳动物视觉发育的关键期前还存在前关键期.目前,前关键期视皮层神经元的反应特性及突触可塑性研究仍处于探索阶段. 目的 探讨小鼠前关键期视皮层神经元的反应特性及突触可塑性特点. 方法 选择生后13～17 d的C57BL/6J小鼠48只,分别采用在体膜片钳全细胞记录及离体脑片膜片钳全细胞记录法记录小鼠视皮层第Ⅳ层神经元的电生理反应.在体记录在小鼠麻醉下进行,在电流钳模式下给予步阶电流刺激,测量其在体膜反应特性.给予最优刺激参数的移动光棒刺激,测量其视觉诱发反应特性.完成在体实验后行离体实验,分别测量神经元离体膜反应特性及白质-第Ⅳ层通路刺激条件下的诱发反应特性.采用随机数字表法将实验动物随机分成4个组,各组雌雄比例分配均匀.每组测定12个细胞,按照刺激频率的不同分别行低频刺激(LFS)和高频刺激[θ波脉冲刺激(TBS)]模式训练,按照刺激时序的不同进行突触前-后(pre-post)模式和突触后-前(post-pre)模式训练,在-70 mV电压钳制下分别记录训练前后兴奋性突触后电流(EPSCs).采用pClmap 10软件对原始数据进行预处理,采用Matlab 2008a软件进行统计分析.结果 在体成功记录的细胞数为39个,离体记录48个.在体和离体条件下视皮层第Ⅳ层神经元稳态平均发放动作电位(AP)个数分别为1.01±0.03和1.01±0.05,AP阈值分别为(-40.2±3.2)mV和(-39.6±2.0)mV,阈电流水平分别为(126.7± 17.4) pA和(129.6±17.5)pA,差异均无统计学意义(AP数:t=0.512,P=0.610;AP阈值:t=-1.074,P=0.286;阈电流:=-0.776,P=0.440).在体最优视觉刺激条件下平均膜电位峰值幅度为(7.3±4.3)mV,鲜见AP;离体最强通路刺激条件下平均膜电位峰值反应幅度为(6.4±2.8)mV,未见AP,在体与离体记录的平均膜电位峰值幅度差异无统计学意义(t=1.234,P=0.221).离体条件下,LFS训练前后EPSCs幅度分别为(138.1±51.9)pA和(76.1±34.8) pA,差异有统计学意义(t=4.437,P=0.001),而TBS训练前后EPSCs幅度差异无统计学意义(t=-0.756,P=0.466),pre-post训练前后EPSCs幅度分别为(122.4±62.2)pA和(78.5±46.7)pA,post-pre训练前后分别为(131.9±48.0)pA和(74.3±30.7)pA,差异均有统计学意义(pre-post:t=3.558,P=0.004;post-pre:t=4.283,P=0.001).结论 前关键期小鼠视皮层第Ⅳ层已完成神经回路的基本构建,但神经元的膜反应性以及突触连接仍未成熟.在低频或高频突触前后时序差异性输入条件下,突触功能受到抑制,而在高频输入条件下突触功能得到继续保持.前关键期小鼠视觉神经系统的发育具有不同于关键期的特征.     

体外培养兔角膜内皮细胞电生理特性的初步研究

目的通过测定体外培养兔角膜内皮细胞(rabbitcorneal endothelial cells,RCECs)静息电位及全细胞电流,初步研究角膜内皮细胞的电生理特性。方法采用后弹力层及内皮层揭取联合组织块法进行RCECs体外培养,采用膜片钳全细胞记录模式记录原代及传代RCECs细胞静息电位及全细胞电流,绘制电流-电压(I/V)曲线。结果体外培养原代RCECs的平均静息膜电位水平为(20.9±1.7)mV(n=6),第2代RCECs的平均静息膜电位水平为(15.1±1.8)mV(n=6),两者差异有统计学意义(P<0.01)。在钳制电位+60mV下,原代RCECs全细胞电流为(396.2±39.0)pA(n=5),大于第2代RCECs的全细胞电流(201.4±45.2)pA(n=5),差异有非常显著的统计学意义(P<0.01)。结论后弹力层及内皮层揭取联合组织块法获得的原代及传代RCECs,在静息电位和全细胞电流等电生理特性方面存在差异。     

睫状神经营养因子对纯化培养大鼠视网膜神经节细胞突起再生的影响

目的观察睫状神经营养因子（ciliary neurotrophic factor，CNTF）对纯化培养大鼠视网膜神经节细胞（retinal ganglion cells，RGCs）突起再生的影响。方法取新生SD大鼠视网膜，消化并利用筛网纯化后接种于96孔板，加入不同浓度（10μg·L-1、20μg·L-1、30μg·L-1、40μg·L-1）CNTF作为实验组，不加CNTF作为对照。根据细胞形态及免疫细胞化学的方法鉴定细胞，观察RGCs生长规律，观测随机选取视野下的RGCs数和有突起细胞数（400倍荧光显微镜和相差显微镜）。结果 纯化的RGCs在含体积分数20％胎牛血清DMEM培养液中可存活30d。加入CNTF后3~5d，40μg·L-1组突起率显著高于对照组（P〈0.05）。7~15d，20μg·L-1、30μg·L-1、40μg·L-1组突起率显著高于时照组（P〈0.05），30μg·L-1、40μg·L-1组突起率显著高于10μg·L-1、20μg·L-1组（P〈0.05）。结论CNTF能显著促进RGCs突起再生，CNTF的浓度和突起率存在量效关系，一定浓度范围内．高浓度CNTF可较早促进突起再生。     

胰高血糖素类肽-1缓释珠对大鼠视网膜神经节细胞的保护作用

目的探讨玻璃体内植入胰高血糖素类肽-1（GLP-1）缓释珠对大鼠视网膜神经节细胞的保护作用。设计实验研究。研究对象SPF级Sprague-Dawley（SD）雄性大鼠25只。方法将25只大鼠随机分为2组,实验组13只,对照组12只。实验组大鼠右眼玻璃体内植入4个GLP-1缓释珠,对照组右眼玻璃体内注入4μl复方氯化钠。GLP-1缓释珠直径600μm,内含3000个整合了GLP-1基因的人骨髓间充质干细胞,外被致密的藻酸盐外膜,以确保GLP-1产物可顺利释放而不引起免疫排斥。玻璃体内注射均在右眼视神经夹伤后立即进行。视神经夹伤后第23天用3％荧光金从双侧上丘做逆行标记,第28天取双眼球标本做视网膜铺片并在荧光显微镜下拍摄照片,采用人工双盲法进行视网膜神经节细胞计数。主要指标视网膜神经节细胞密度以及视网膜神经节细胞存活率。结果视网膜神经节细胞密度实验组与对照组分别为（2113±474）／mm2和（1734±424）／mm2,两组之间的差异有统计学意义（t=2．111,P=-0．046）。视网膜神经节细胞存活率实验组与对照组分别为（74±18）％和（57±16）％,两组之间的差异有统计学意义（t=-2．451,P=-0．022）。结论GLP-1缓释珠玻璃体内植入后对视神经夹伤大鼠视网膜神经节细胞具有保护作用,可以提高视网膜神经节细胞存活率。     

17β-雌二醇对高氧诱导的鼠视网膜新生血管形成的影响

目的 观察并探讨17β-雌二醇对高氧诱导的鼠视网膜新生血管形成的影响及机制。方法 48只新生Sprague-Dawley(SD)大鼠随机分为对照组和实验组,每组各24只。对照组大鼠分娩完成后与新生鼠一起正常饲养;实验组大鼠分娩完成后立即与新生鼠一起置于高氧环境饲养。对照组和实验组又再分为磷酸盐缓冲液（PBS）干预组（对照组1、实验1）和雌二醇干预组（对照组2、实验组2),每组各12只大鼠。对照组1和实验组1大鼠分别每日皮下注射PBS 0.1 ml;对照组2和实验组2大鼠分别每日皮下注射雌二醇 1 μg。每日观察鼠的发育情况。出生后7、14 d逆转录聚合酶链反应(RT PCR)检测视网膜血管内皮生长因子(VEGF)、低氧诱导因子-1α（HIF-1α）mRNA的含量。出生后14 d时行苏木精 伊红（HE）染色观察视网膜新生血管生长情况;免疫组织化学染色观察VEGF蛋白的表达;透射电子显微镜观察视网膜超微结构变化。结果出生后14 d各组大鼠标本中突破内界膜的内皮细胞数比较,差异有统计学意义（F=10.7,P＜0.05）。其中,实验组1较对照组1明显增多,差异有统计学意义（q=4.28,P＜0.05）。实验组2较实验组1明显减少,差异有统计学意义（q=5.16,P＜0.05）。实验组2与对照组1比较,差异无统计学意义（q=0.25,P＞0.05）。出生后14 d各组大鼠VEGF蛋白表达比较,差异有统计学意义（F=10.7,P＜0.05）。其中,实验组1与对照组、实验组2比较,差异有统计学意义（q=5.41,4.35,P＜0.05）。视网膜超微结构显示,实验组1神经节细胞肿胀,细胞质淡染,线粒体空泡形成;其他各组视网膜超微结构正常。出生后7、14 d各组大鼠视网膜VEGF、HIF-1 mRNA表达比较,差异均有统计学意义（F=14.7,16.1,13.4,17.5;P=0.001,0.005,0.003,0.009）。其中,出生后7 d,对照组2 VEGF表达高于对照组1,实验组2VEGF表达高于实验组1,组间比较,差异均有统计学意义（q=5.22,4.32;P＜0.05）。出生后14 d,对照组2 VEGF表达高于对照组1,实验组2 VEGF、HIF-1表达低于实验组1,组间比较,差异均有统计学意义（q=3.72,5.12,4.08;P均＜0.05）。结论雌二醇对VEGF mRNA具有双重调控作用,高氧条件下促进视网膜VEGF表达和视网膜血管发育;正常氧条件下通过HIF-1α-VEGF系统抑制视网膜新生血管形成。雌二醇在一定程度上可保护缺氧造成的视网膜超微结构损害。     

Photoreceptor calcium channels: insight from night blindness

The genetic locus for incomplete congenital stationary night blindness (CSNB2) has been identified as the CACNA1f gene, encoding the alpha 1F calcium channel subunit, a member of the L-type family of calcium channels. The electroretinogram associated with CSNB2 implicates alpha 1F in synaptic transmission between retinal photoreceptors and bipolar cells. Using a recently developed monoclonal antibody to alpha 1F, we localize the channel to ribbon active zones in rod photoreceptor terminals of the mouse retina, supporting a role for alpha 1F in mediating glutamate release from rods. Detergent extraction experiments indicate that alpha 1F is part of a detergent-resistant active zone complex, which also includes the synaptic ribbons. Comparison of native mouse rod calcium currents with recombinant alpha 1F currents reveals that the current-voltage relationship for the native current is shifted approximately 30 mV to more hyperpolarized potentials than for the recombinant alpha 1F current, suggesting modulation of the native channel by intracellular factors. Lastly, we present evidence for L-type alpha 1D calcium channel subunits in cone terminals of the mouse retina. The presence of alpha 1D channels in cones may explain the residual visual abilities of individuals with CSNB2.     

利用锥体细胞失功能大鼠和先天性静止性夜盲大鼠对视锥与视杆通路振荡电位的分离研究

背景 振荡电位(OPs)是评估视网膜缺血缺氧性疾病视网膜功能变化的重要工具,利用视网膜退行性病变动物模型对视锥、视杆通路起源的OPs特点进行研究非常重要. 目的 在两种自发性视网膜退行性病变模型大鼠中分离视锥、视杆通路,对比分析视杆、视锥通路起源的OPs波的特点. 方法 采用雄性SD大鼠、锥体细胞失功能(RCD)大鼠、先天性静止性夜盲(CSNB)大鼠各6只,以RETI-scan视觉生理记录系统分别在暗适应(12h)和明适应(10 min)条件下,用不同强度的刺激光(-35、-25、-15、-5、0、5 db)进行刺激,记录各组大鼠的闪光视网膜电图(FERG),通过Matlab 7.0的Butterworth滤波提取OPs,采用快速傅里叶变换(FFT)对所得OPs进行频谱分析.结果 暗适应条件下SD大鼠和RCD大鼠的ERG均可见a波和b波,但CSNB大鼠b波阙如;明适应条件下,SD大鼠和CSNB大鼠可见b波,但RCD大鼠各波阙如.暗适应较高刺激光强度下,SD大鼠和RCD大鼠均有低频(主频)和高频(次频)两个明显的频峰,分别为75 ～ 110 Hz、90～120 Hz和90～ 120 Hz、110 ～ 135 Hz;不同刺激光强度下,CSNB大鼠只有一个频峰,为70～100 Hz.而明适应不同刺激光强度下,SD大鼠和CSNB大鼠均只有一个频峰,分别为75～95 Hz和70～85 Hz.明适应条件下与SD大鼠比较,CSNB大鼠b波隐含时延长,b波振幅明显下降,差异均有统计学意义(P＜0.05);暗适应条件下,RCD大鼠b波隐含时和振幅与SD大鼠比较,差异无统计学意义(P＞0.05);与SD大鼠比较,RCD和CSNB大鼠OPs波振幅下降,隐含时延长,差异均有统计学意义(P＜0.05);明适应条件下不同刺激光强度下CSNB大鼠OPs波的隐含时明显长于SD大鼠,振幅明显低于SD大鼠,差异均有统计学意义(P＜0.05). 结论 视锥、视杆通路起源的OPs有不同特性,自发性视网膜退行性改变大鼠的视杆OPs有两个频峰,正常情况下,视杆通路对OPs的贡献比视锥通路大.     

Voltage-gated calcium and sodium currents of starburst amacrine cells in the rabbit retina

The voltage-gated calcium and sodium currents of starburst amacrine cells were examined in slices of the adult rabbit retina. ON-center starburst amacrine cells were targeted for whole-cell recording by prelabeling the retina with the nuclear dye 4'-6-diamidino-2-phenylindole hydrochloride (DAPI). Calcium currents were isolated using an external Ringer that contained tetrodotoxin to block sodium currents and barium to block potassium channels. When starburst amacrine cells were stepped to holding potentials positive to -50 mV, a series of voltage-dependent calcium currents were activated. The calcium current peaked at -10 mV. The calcium currents kinetics were mainly sustained in nature, showing only a small amount of slow inactivation. Nickel (100 microM), a T-type channel blocker, had no effect on the calcium current. Application of the L-type channel agonist BAY K8644 (1-2.5 microM) had small variable effects on the calcium current while the L-type channel antagonist nifedipine (10 microM) had no effect. However, addition of a reported N-type calcium channel antagonist, omega-conotoxin G6A (1 microM), blocked a large portion of the calcium current, as did a more nonselective antagonist, omega-conotoxin M7C (200 nM). Agatoxin 4A (500 nM) reduced a smaller sustained calcium current component, implying a P/Q-type calcium channel was present on these neurons. In addition to the calcium currents, a fast voltage-gated sodium current was observed in many starburst cells. This current could be blocked by tetrodotoxin (200-500 nM). The differing kinetics and durations of the sodium and calcium currents could play important roles in the regulation of synaptic release and in the coordination of spiking by starburst amacrine cell dendrites during retinal development and in the encoding of motion across the retinal surface.     

N-type and L-type calcium channels mediate glycinergic synaptic inputs to retinal ganglion cells of tiger salamanders

Synaptically localized calcium channels shape the timecourse of synaptic release, are a prominent site for neuromodulation, and have been implicated in genetic disease. In retina, it is well established that L-type calcium channels play a major role in mediating release of glutamate from the photoreceptors and bipolar cells. However, little is known about which calcium channels are coupled to synaptic exocytosis of glycine, which is primarily released by amacrine cells. A recent report indicates that glycine release from spiking AII amacrine cells relies exclusively upon L-type calcium channels. To identify calcium channel types controlling neurotransmitter release from the population of glycinergic neurons that drive retinal ganglion cells, we recorded electrical and potassium evoked inhibitory synaptic currents (IPSCs) from these postsynaptic neurons in retinal slices from tiger salamanders. The L-channel antagonist nifedipine strongly inhibited release and FPL64176, an L-channel agonist, greatly enhanced it, indicating a significant role for L-channels. omega-Conotoxin MVIIC, an N/P/Q-channel antagonist, strongly inhibited release, indicating an important role for non-L channels. While the P/Q-channel blocker omega-Aga IVA produced only small effects, the N-channel blocker omega-conotoxin GVIA strongly inhibited release. Hence, N-type and L-type calcium channels appear to play major roles, overall, in mediating synaptic release of glycine onto retinal ganglion cells.     

Voltage-activated Ca2+ channels and ionotropic GABA receptors localized at axon terminals of mammalian retinal bipolar cells.

A preparation of isolated presynaptic terminals of rat retinal rod bipolar cells was developed. Patch-clamp recordings were performed on the isolated terminal to determine the type(s) of voltage-activated Ca2+ channels and the contribution of GABA(A) and GABA(C) receptor-mediated currents localized in the terminal region. Both low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca2+ currents, with properties similar to those found in intact cell recordings, were observed in the isolated terminal recordings. Consistent with previous studies, the HVA Ca2+ currents are L-type since the currents were blocked by low micromolar concentrations of nimodipine and potentiated by BayK 8644. Also, both GABA(A) and GABA(C) receptor-mediated currents were observed in the isolated terminal. The current density of GABA(C) receptors in the terminal was more than three times higher than that in the soma. In contrast, the current density of GABA(A) currents between the terminal and the soma was not significantly different. Assessed by 100 microM GABA, the contributions of GABA(A) and GABA(C) receptors to the total GABA-mediated currents at the terminal were comparable. This study directly demonstrates the localization of LVA Ca2+ channels at the axon terminal of mammalian rod bipolar cells, suggesting that LVA Ca2+ channels may play a role in bipolar cell transmitter release. Results of this study also support the notion that both types of ionotropic GABA receptors regulate synaptic transmission in mammalian rod bipolar cells. In addition, this study reports for the first time the feasibility of direct patch-clamp recordings of isolated axon terminals of mammalian retinal bipolar cells. The isolated presynaptic terminal preparation of mammalian retinal bipolar cells could be a valuable system for the study of transmitter release in the central nervous system (CNS).     

视网膜动脉平滑肌细胞中大电导钙离子激活钾通道电流和钙离子浓度变化对糖尿病视网膜动脉收缩的影响

背景 糖尿病视网膜病变(DR)是常见的视网膜微血管并发症,视网膜血管平滑肌细胞大电导钙激活钾离子通道(BK)是调节血管舒缩和血液动力的主要因素.目前关于视网膜动脉平滑肌细胞(RASMCs)上BK通道的功能变化在DR形成中的作用鲜有研究报道. 目的 研究正常及糖尿病模型大鼠RASMCs中BK通道电流、钙离子浓度及不同钙离子浓度下BK通道开放概率(NP0)的变化,探讨DR早期的血管损伤机制. 方法 采用随机数字表法将50只SPF级8～12周龄SD大鼠随机分为正常对照组和糖尿病模型组,糖尿病模型组40只大鼠腹腔内注射60 mg/kg链脲佐菌素(STZ)法制作1型糖尿病模型,正常对照组大鼠10只同法注射枸橼酸钠溶液.采用酶消化法分离大鼠RASMCs,应用全细胞膜片钳技术记录大鼠RASMCs中BK通道电流;采用荧光探针法测定大鼠RASMCs内钙离子浓度;采用膜内向膜外型单通道膜片钳技术记录不同钙离子浓度条件下RASMCs中BK单通道NP0的变化.结果 36只大鼠糖尿病造模成功,造模成功率为90％.刺激电压大于60 mV时,糖尿病模型组大鼠RASMCs中BK通道电流密度明显下降,刺激电压为100 mV时,正常对照组和糖尿病模型组大鼠RASMCs BK通道电流分别为(100±23) PA/PF和(50±7) PA/PF,差异有统计学意义(t=19.80,P＜0.05).当加入BK通道特异性阻滞剂非洲蝎毒素100 nmol后,正常对照组的BK通道电流明显减弱,而糖尿病模型组BK通道电流无明显变化;正常对照组和糖尿病模型组大鼠RASMCs内钙离子浓度分别为(123±11)nmol/L和(255士10)nmol/L,差异有统计学意义(t=32.50,P＜0.05).在刺激电位为60 mV条件下,随着钙离子浓度增加,BK通道NP0增加,差异有统计学意义(F=15.28,P＜0.05).结论 糖尿病模型大鼠RASMCs中BK通道电流下降,细胞内钙离子浓度升高,BK单通道NP0下降.BK通道功能改变可能是导致糖尿病时视网膜动脉异常收缩的主要原因之一.     

Functional modifications in rod bipolar cells in a mouse model of retinitis pigmentosa

The rd mouse has been widely used as an animal model of retinitis pigmentosa. In this model, a mutation of rod-specific phosphodiesterase leads to a loss of rods during the early period of postnatal life. Morphological modifications at the level of the outer plexiform layer have been shown (Proc. Nat. Acad. Sci. USA 97 (2000) 11020) in bipolar and horizontal cells. However, very little is known about the functional changes suffered by these cells postsynaptic to the degenerated rods. In the present work we have studied the neurotransmitter-induced currents in rod bipolar cells from the rd mouse retina. Currents induced by glutamate and GABA were studied by the patch clamp-whole cell technique, on rod bipolar cells enzymatically dissociated from the rd mouse retina. Data from rd animals were compared with non-dystrophic NMRI mice. GABA (30-100 micro M) and glutamate (100 micro M) were applied from a puff pipette in the near proximity of rod bipolar cell dendrites, clamped at physiological membrane potentials, and their evoked currents were studied. In rod bipolar cells from non-dystrophic mouse, puff application of glutamate induced an outward current. This current was increased twofold in absence of extracellular calcium (nominally 0 calcium). In rod bipolar cells from adult rd mouse, currents induced by glutamate were absent. Two types of GABA mediated currents were isolated in rod bipolar cells both in control and rd mouse retinas. The currents mediated by GABA(C) receptors were observed exclusively at the axon terminal, while the currents mediated by the GABA(A) receptors were observed upon GABA application to the bipolar cell dendrites. The currents mediated by GABA(A) receptors in rod bipolar cells from rd mouse were larger than those from control animals. We conclude that after the degeneration of rod photoreceptors in rd mouse, rod bipolar cells lost their glutamate (rod-neurotransmitter) input while they increase their response to GABA (horizontal cell-neurotransmitter). In our opinion, this work describes for the first time the changes in neurotransmitter sensitivity that affect rod bipolar cells after photoreceptor degeneration of the mouse retina.