重组人促红细胞生成素对离体培养视网膜神经节细胞的影响

目的研究重组人促红细胞生成素（recombinant humanerythropoiain，rhEPO）对培养的视网膜神经节细胞（retinal ganglion cells，RGCs）存活、突起生长及生长相关蛋白43（growth associmed protein43，GAP-43）表达的影响，探讨rhEPO对培养RGCs可能的作用机制。方法分别用DMEM及含有rhEPO的DMEM对RGCs进行离体培养，观察RGCs在体外存活的时间，测量2d、4d、6dRGCs的最长突起长度；免疫细胞化学检测GAP-43蛋白表达，并测定平均灰度值。结果DMEM组离体培养RGCs于6～8d死亡，而rhEPO组RGCs能存活10～12d，存活时间较DMEM组显著延长（P〈0．01）。培养2d、4d、6d时，DMEM组最长突起长度依次为（42．90±4．71）μm、（79．74±8．49）μm、（110．02±10．79）μm，rhEPO组依次为（55．47±7．07）μm、（100．16±7．78）μm、（118．63±11．50）μm，培养2d、4d时rhEPO组与DMEM组相比差异非常显著（P〈0．01），6d时差异显著（P〈0．05）。2组细胞在培养2d时GAP-43蛋白的表达水平较高，4d时GAP-43蛋白表达到高峰。6d时GAP-43蛋白的表达水平明显降低。各时间点rhEPO组RGCsGAP-43蛋白的表达均较DMEM组有明显增高，与DMEM组相比均有非常显著差异（P〈0．01）。结论rhEPO可延长离体培养RGCs的存活时间和促进其突起的生长。上调离体培养RGCs GAP-43蛋白的表达。rhEPO促进RGCs突起生长的作用可能通过上调RGCs GAP-43蛋白的表达来实现。[眼科新进展2007；27（3）：138-192]     

天麻钩藤饮抗大鼠视神经夹伤模型视网膜神经节细胞凋亡作用的实验研究

目的 观察天麻钩藤饮对视神经夹伤模型大鼠视网膜神经上皮层厚度和视网膜神经节细胞（RGC）凋亡的影响。设计 实验研究。研究对象 SPF级雄性Wistar大鼠48只。方法 将大鼠随机分6组,每组8只,分别为正常对照组,阴性对照组,天麻钩藤饮低剂量组（0.6 g/ml）、中剂量组（1.2 g/ml）、高剂量组（2.4 g/ml）和银杏叶片阳性对照组（1.2 mg/ml）,除正常对照组大鼠不做处理,其他组大鼠右眼均建立视神经夹伤模型,正常对照组和阴性对照组给予纯净水灌胃。于给药30天处死动物取眼球,做石蜡切片HE染色,观察各组视网膜神经上皮层厚度,TUNEL法检测RGC的凋亡程度。主要指标 HE染色视网膜神经上皮厚度及RGC凋亡数量。结果 阴性对照组（171.04±13.86 μm）比正常组（208.98±8.46 μm）视网膜神经上皮厚度显著减少（P=0.000）。而天麻钩藤饮中、高剂量组大鼠视网膜厚度（分别为187.68±11.16 μm 和189.22±9.54 μm）比阴性对照组显著增加（P=0.043,0.001）,且中、高剂量组与阳性对照组（191.35±9.03 μm）之间无显著差异（P=0.052,0.670）;TUNEL凋亡检测发现,阴性对照组凋亡细胞数（9.09±2.24个/高倍视野）比正常组（0.59±0.61个/高倍视野）明显增加（P=0.000）,中剂量组、高剂量组和阳性对照组RGC的凋亡（分别为7.00±1.88, 5.22±2.05, 5.03±2.03个/高倍视野）均比阴性对照组显著减少（P=0.024, 0.000,0.000）。结论 天麻钩藤饮对大鼠视神经夹伤模型具有一定抗RGC凋亡的作用,随药物浓度的升高,抗凋亡作用更显著。     

Role of extracellular signal-regulated kinase in glutamate-stimulated apoptosis of rat retinal ganglion cells

PURPOSE: To investigate the involvement of the extracellular signal-regulated kinase (ERK) signaling pathway after intravitrevous injection of glutamate in rat retina. METHODS: Three groups of five Sprague-Dawley rats each were studied. Group I was a normal control group, intravitreal saline injections. In Group II, one eye received an intravitreal glutamate injection (375 nmol, dissolved in saline) while the contralateral eye served as control. In Group III, intravitreal PD98059 (100 micro mol, an inhibitor of ERK) injections were administered 1 hr before glutamate injections. Seven days after injections, phosphorylated (activated) ERK in retina was localized by immunohistochemistry and fluorescent double labeling of retinal cryosections. Specific ERK blockade was documented to assess the functional significance of activated ERK. TUNEL staining was performed to assess apoptotic cell death. RESULTS: Expression of phosphorylated ERK in rat retina was observed in the inner nuclear layer, the outer nuclear layer, and the nerve fiber layer after 3 days intravitreous injection of glutamate, increasing significantly after 7 days. Double immunofluorescence labling demonstrated that the increased retinal immunostaining for phospho-ERK was predominantly localized to the retinal Müller cells after 7 days intravitreous injection of glutamate. Moreover, blocking activation of ERK significantly improved the number of TUNEL-positive cells in the eyes receiving intravitreal PD98059 injections compared with the eyes receiving glutamate injections. CONCLUSIONS: The ERK pathway is involved in signal transduction in the retina after excessive stimulation by glutamate, which may contribute to the antiapoptotic role in retinal ganglion cell death induced by glutamate.     

不同强度微波辐射对SD大鼠视网膜神经节细胞凋亡的影响

目的观察不同强度微波辐射对视网膜神经节细胞（retinal ganglion cells，RGcs）凋亡的影响。方法体外培养RGCs，经频率为2450MHz微波辐射1h，按辐射强度分为4组：0mW·cm^-2组、10mW·cm^-2组、30mW·cm^-2组和60mW·cm^-2组。组。倒置显微镜下观察细胞形态。台盼蓝染色检测细胞存活率，Annexin V—PI荧光标记双染色流式细胞术和TUNEL法检测RGCs的凋亡率。结果各组细胞经辐射后形态均有所改变，细胞存活率分别为98．7％、98．4％、87．8％和63．8％，随微波强度增加而下降；而流式细胞术检测的细胞凋亡率的结果表现出强度及时间相关性，TUNEL法检测的细胞凋亡率分别为5．2％、12．2％、18.0％和18.5％，随微波强度增加而增加。结论2450MHz微波可引起RGCs损伤及细胞凋亡，其损伤程度表现出辐射剂量相关性。     

Tafluprost protects rat retinal ganglion cells from apoptosis in vitro and in vivo

Background

To investigate whether tafluprost, which is a prostaglandin-related compound and an anti-glaucoma drug, has a direct anti-apoptotic effect in cultured retinal ganglion cells (RGCs) and rat RGCs in retinas with optic nerve crush (ONC).

Methods

RGC-5 cells were induced to undergo apoptosis by a serum deprivation and by exogenous glutamate. The level of cell death with or without tafluprost was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in intracellular calcium ([Ca²⁺]i) levels were measured with fluo-4 fluorescence. Rat RGCs were degenerated by ONC. After topical instillation of tafluprost for 7 and 14 days, the numbers of retrograde-labeled RGCs were counted. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells.

Results

Tafluprost dose-dependently promoted RGC-5 cell viability with an optimum concentration of 3?μM (p?=?0.006). Tafluprost significantly reduced caspase-3-positive cells and suppressed [Ca⁺²]i evoked by exogenous glutamate. The cGMP-dependent protein kinase inhibitor and KT-5823 partially blocked the rescue effect of tafluprost (p?=?0.002). The survival rate of RGCs significantly increased in eyes treated with tafluprost (p?=?0.01), and the prevalence of TUNEL-positive cells was significantly decreased 14 days after ONC (p?<?0.001).

Conclusions

These data suggest that tafluprost has an anti-apoptotic effect in RGCs.     

Latanoprost protects rat retinal ganglion cells from apoptosis in vitro and in vivo

We investigated whether latanoprost has a direct anti-apoptotic effect in retinal ganglion cell (RGC) line and RGCs in the rat. RGC-5 cells were induced to undergo apoptosis by serum deprivation and exogenous glutamate. The level of cell death with or without latanoprost acid was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in the level of intracellular calcium ([Ca²⁺]i) were measured with fluo-4 fluorescence. The XTT assay revealed that latanoprost acid increased RGC-5 cell viability. Latanoprost acid significantly reduced caspase-3 positive cells and suppressed [Ca²⁺]i evoked by glutamate. U0126, a mitogen-activated protein/extracellular signal-regulated kinase 1 and 2 inhibitor, partially blocked the rescue effect of latnanoprost acid (p = 0.013). In vivo, rat RGCs were degenerated by optic nerve crush. After topical instillation of latanoprost for 7 days, RGCs labeled with fluorogold were significantly. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells. TUNEL-positive cells were significantly decreased in eyes with topically instilled latanoprost (p = 0.015). These data suggest that latanoprost has an neuroprotective ability in RGCs.     

视网膜下移植睫状神经营养因子编码基因修饰的细胞保护SD大鼠视神经横断伤后视网膜节细胞变性

目的探讨移植表达睫状神经营养因子(CNTF)的细胞对SD大鼠视神经横断伤后视网膜节细胞的保护作用。方法通过脂质体将CNTF表达质粒转移至人胚肺成纤维细胞,建立稳定、高水平表达CNTF的细胞株。采用双侧背外侧膝状体及上丘核团注射3%荧光金逆行标记视网膜节细胞。将标记后的大鼠分为两组,于标记后7d手术切断眶内段视神经其中一组左眼不做手术作为正常对照组,右眼切断视神经作为手术对照组;另一组双眼均手术切断视神经,左眼注射PBS作为治疗对照组,右眼视网膜下移植表达CNTF的细胞作为实验组。术后5、14、17、21及28d取出眼球,铺片后荧光显微镜观察并计数视网膜内存活的节细胞。结果手术切断眶内段视神经后2周,视网膜内节细胞数减少6744%,视网膜下移植表达CNTF的细胞后第5、17、21d视网膜内存活的节细胞数明显多于治疗对照组(P<005)。结论视网膜下移植高水平表达CNTF的细胞对视网膜节细胞有保护作用。     

Intravitreal administration of erythropoietin and preservation of retinal ganglion cells in an experimental rat model of glaucoma

PURPOSE: The aim of this pilot study was to evaluate the potential neuroprotective effect of an intravitreal injection of erythropoietin (EPO) on retinal ganglion cell (RGC) preservation in an episcleral vessel cautery-induced rat model of glaucoma. METHODS: The animals were randomly assigned into an unoperated control group (n = 11) and three experimental groups: episcleral vessel cautery only (EVC: n = 4), episcleral vessel cautery with intravitreal normal saline injection (EVC-NS; n = 5), and episcleral vessel cautery with intravitreal EPO treatment (EVC-EPO; n = 9). The intravitreal injections were limited to 5 mul containing either normal saline alone or 200 ng of EPO in normal saline administered immediately after the cautery procedure. RGCs were labeled retrogradely by FluoroGold neuron tracer 5 to 7 days prior to the collection of eyes at day 21 and counted in whole flat-mounted retinas with fluorescence microscopy. RESULTS: Compared to the RGC counts in retinal specimens from unoperated control rats (12,619 +/- 310), the corresponding RGC counts were significantly decreased in both the EVC (9116 +/- 273; p < 0.005) and EVC-NS (9489 +/- 293; p < 0.005) groups but not significantly decreased in the EVC-EPO (11,212 +/- 414; p = 0.051) treated retinas. CONCLUSIONS: A single intravitreal 200 ng dose of EPO appears to have a protective effect on RGC viability in an in vivo rat model of glaucoma. Further experimental studies are needed to confirm these preliminary results and to optimize the appropriate dose and frequency of EPO delivery in animal models of glaucoma.     

Effects of retinal glial cells on isolated rat retinal ganglion cells

PURPOSE: The effect of retinal glial cells on retinal ganglion cell (RGC) survival was investigated in cocultures of pure, isolated retinal glial cells with pure, isolated RGCs. METHODS: RGCs from 2-day-old rats were cocultured for 48 hours, avoiding direct contact between cell types, with either nonconfluent retinal glial cells from 3-day-old rats or confluent retinal glial cells from 3-day-old, 12-day-old, or 1-year-old rats. Survival of RGCs was evaluated by flow cytometry. Amino acids were determined in culture medium. The effects of glutamate antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione and MK801, a nitric oxide (NO) scavenger, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), and an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), were examined. RESULTS: Nonconfluent retinal glial cells significantly reduced the survival of small and large RGCs, but confluent retinal glial cells reduced the survival of only small RGCs, regardless of the rat's age at the time of retinal glial cell harvesting. Profiles of some amino acids significantly varied, depending on the culture condition. Cocultures of RGCs with nonconfluent retinal glial cells released significantly more glutamate into the medium than cocultures of RGCs with confluent retinal glial cells or RGCs in pure culture. The glutamate antagonists improved the survival of RGCs cocultured with nonconfluent retinal glial cells, especially when the two were administered in combination, and in the case of large RGCs. c-PTIO and L-NAME, also improved the survival of RGCs cocultured with nonconfluent retinal glial cells. CONCLUSIONS: Adverse effects of retinal glial cells on the survival of RGCs varied by size of the RGCs and retinal glial cell confluence. Glutamate and NO may be involved in retinal glial cell-related antisurvival effects.     

促红细胞生成素对视网膜神经节细胞保护作用的研究进展

青光眼是一组由病理性眼压升高导致进行性视网膜神经节细胞凋亡和视野缺损的眼病,通过延缓视网膜神经节细胞（retinal ganglion cells,RGC）凋亡进度对受损视神经进行保护是青光眼研究领域的重要方向。近几年在对视神经保护的药物研究中,促红细胞生成素（erythropoietin, EPO）受到广泛关注。EPO 通过与红细胞膜表面的促红细胞生成素受体（erythropoietin receptor, EPOR）结合,抑制不同的细胞信号转导通路（如 HIF-1／iNOS 通路、RhoA ／ROCK 通路等）,从而抑制细胞内诱导凋亡发生的 Bax／Bcl-2等复合物的形成,发挥视神经保护作用。（国际眼科纵览,2016,40：155-160）     

Cytoarchitecture of the retinal ganglion cells in the rat

PURPOSE: To determine the number and cytoarchitecture of retinal ganglion cells (RGCs) in the female Wistar rat, by using a newly devised procedure for rapid RGC counting in the entire retina that avoids assumptions about RGC spatial arrangement. METHODS: RGCs of normal female Wistar rats were retrogradely labeled with a fluorescent tracer. Automated counting was accomplished by applying standard imaging software to analysis of all labeled cells in retinal flatmounts. The method was validated by comparison of automated and manual counts of 70,000 RGCs in frames covering the density range in the normal rat retina of 600 to 3600 RGC/mm(2). RGC numbers were determined for each retina and compared with the contralateral retina of the same animal. RGC density maps were constructed for each retina. RGC size distribution was determined. RESULTS: Automated RGC counting showed a good linear correlation with manual counting (R(2) = 0.9416). Mean total RGC count in 10 rat eyes was 97,609 +/- 3,930 (SEM) per eye. Contralateral eyes differed by an average of 4.1% (3983 plus minus 5098 RGCs). Size analysis calculated from cell areas confirmed that the majority of rat RGCs are between 7 and 21.5 microm in equivalent diameter. The RGC counts for all frames at the same eccentricity in all 10 of the retinas showed that variability increased with eccentricity and increased further as the fractional area of the retina sampled at each eccentricity was reduced. There was also significant variability in the spatial density of the RGCs at the same eccentricity location between different eyes. Comparison of total RGC counts between left and right eyes estimated from RGC counts in sectors of the retina (hemiretinas or quadrants) showed increased variability compared with counting all the RGCs in a retina. CONCLUSIONS: RGCs in the Wistar rat display significant variability in their cytoarchitecture. Such variability can make quantification by sampling problematic for diffuse, and particularly, for focal RGC losses resulting from experimental interventions, unless virtually the entire RGC population is counted.     

促红细胞生成素缓释微球玻璃体腔注射对视网膜神经节细胞的保护作用

目的 探讨乳酸/羟基乙酸共聚物（PLGA）装载的促红细胞生成素（EPO）缓释微球（EPO-PLGA微球）经玻璃体腔注射对大鼠视神经挫伤模型中受损视网膜神经节细胞（RGC）的保护作用.方法 选取成年SD大鼠,建立视神经挫伤模型.建模后分别经玻璃体腔内注射含10 IU EPO的PLGA微球（EPO-PLGA组）、10 IU EPO（EPO组）、5 μl空白PLGA（PLGA组）、5 μl PBS（PBS组）,另设未治疗组不予玻璃体腔注药.术后5 d和2周,做视网膜切片,对各组RGC凋亡情况行TUNEL检测;术后23 d,DiI上丘逆标RGC,并于术后4周处死大鼠,视网膜铺片观察各组RGC存活情况;每组各个时间点分别处死6只SD大鼠.采用方差分析对结果进行比较.结果 TUNEL检测显示,术后5 d和2周,各组均可见TUNEL阳性细胞,其中EPO-PLGA组和EPO组TUNEL阳性细胞显著减少,其细胞凋亡率明显少于PLGA组、PBS组及未治疗组.术后4周,视网膜铺片RGC计数显示,正常SD大鼠RGC密度为（2387.7±164.9）个/mm^2,未治疗组为（748.3±58.8）个/mm^2,EPO-PLGA组为（1296.7±157.6）个/mm^2,EPO组为（1418.5±154.9）个/mm^2,PLGA组为（821.7±52.1）个/mm^2,PBS组为（804.4±86.4）个/mm^2;可见EPO-PLGA组和EPO组较未治疗组细胞密度显著增高,具有明显的RGC保护作用（P均＜0.01）,而EPO-PLGA组和EPO组间差异无统计学意义（P=0.065）.结论 EPO-PLGA缓释微球与EPO具有等效的RGC保护作用,这为进一步观察EPO-PLGA缓释微球的长效神经保护作用奠定了基础.     

Large-scale morophological survey of rat retinal ganglion cells

Ganglion cells in an isolated wholemount preparation of the rat retina were labeled using the "DiOlistic" labeling method (Gan et al., 2000) and were classified according to their morphological properties. Tungsten particles coated with a lipophilic dye (DiI) were propelled into the wholemount retina using a gene gun. When a dye-coated particle contacted the cell membrane, the entire cell was labeled. The ganglion cells were classified into four types based on their soma size, dendritic-field size, branching pattern, and level of stratification. Broadly monostratified cells were classified into three types: RG(A) cells (large soma, large dendritic field); RG(B) cells (small- to medium-sized soma, small- to medium-sized dendritic field); and RG(C) cells (small- to medium-sized soma, medium-to-large dendritic field). Bistratified cells were classified as RG(D). Several subtypes were identified within each ganglion cell group. A number of new subtypes were discovered and added into the existing catalog, among them were two types of bistratified cells. This study therefore represents the most complete morphological classification of rat retinal ganglion cells available to date.     

The maintained discharge of rat retinal ganglion cells

Action potentials were recorded from rat retinal ganglion cell fibers in the presence of a uniform field, and the maintained discharge pattern was characterized. Spike trains recorded under ketaminexylazine. The majority of cells had multimodal interval distributions, with the first peak in the range of 25.00.97). Both ON and OFF cells show serial correlations between adjacent interspike intervals, while ON cells also showed second-order correlations. Cells with multimodal interval distribution showed a strong peak at high frequencies in the power spectra in the range of 28.9-41.4 Hz. Oscillations were present under both anesthetic conditions and persisted in the dark at a slightly lower frequency, implying that the oscillations are generated independent of any light stimulus but can be modulated by light level. The oscillation frequency varied slightly between cells of the same type and in the same eye, suggesting that multiple oscillatory generating mechanisms exist within the retina. Cells with high-frequency oscillations were described well by an integrate-and-fire model with the input consisting of Gaussian noise plus a sinusoid where the phase was jittered randomly to account for the bandwidth present in the oscillations.     

促红细胞生成素对大鼠实验性视网膜脱离细胞凋亡的干预作用

目的探讨促红细胞生成素(erythropoietin,EPO)对实验性视网膜脱离大鼠细胞凋亡的干预作用。方法选用SD大鼠44只,随机分为正常对照组(4只)、生理盐水(normal saliue,NS)对照组20只和EPO治疗组(20只)。正常对照组不做任何处理,其他两组大鼠建立孔源性视网膜脱离模型。造模前4hEPO治疗组大鼠腹腔中注射EPO,NS对照组注射生理盐水。术后1h、6h、12h、24h、72h处死大鼠,每个时间点各取4只,摘取眼球,TUNEL染色,计算内核层、节细胞层及外核层细胞数,进行统计学分析。结果正常对照组大鼠视网膜层次结构清晰,各层排列整齐。NS对照组在脱离后12h连续层状结构紊乱,外节丢失增多,而EPO治疗组的连续层状结构存在,外节变性较轻。TUNEL染色结果显示EPO治疗组在视网膜脱离后1h、12h、24h、72h节细胞层及内核层凋亡细胞数[分别为(4.75±1.42)/目、(7.17±1.27)/目、(9.41±1.08)/目、(11.83±1.11)/目]少于NS对照组(7.83±1.70)/目、(9.67±1.56)/目、(12.42±1.31)/目、(15.25±1.22)/目,差异有统计学意义(均为P<0.001)。而外核层EPO治疗组在视网膜脱离后12h、24h、72h的凋亡细胞数[分别为(33.08±8.64)/mm2、(49.17±12.52)/mm2、(92.83±14.14)/mm2]少于NS对照组,差异有统计学意义(均为P<0.05),余时段2组间比较差异均无统计学意义。结论通过腹腔注射EPO能减少实验性视网膜脱离所致视网膜各层细胞的凋亡。     

视网膜神经节细胞凋亡的信号传导与基因调控

青光眼视功能损害的病理基础是视网膜神经节细胞（RGCs)的进行性死亡和视神经纤维的丢失，神经节细胞死亡主要是通过细胞凋亡的方式进行的。刺激RGCs凋亡的信号有多种，如视网膜缺血，兴奋性谷氨酸释放，一氧化氮（NO）增多，神经营养因子剥夺及其他细胞外环境的改变。这些细胞外部因素通过不同的信号传导途径影响着细胞内控制凋亡的相关基因，如caspase-3,bcl-2及p53，从而间接地调控细胞凋亡。从视网膜神经节细胞凋亡的信号传导途径和细胞凋亡的基因调控两方面介绍其研究进展。并探求其可能的保护机制。     

分泌型糖蛋白Reelin对N-甲基-D-天冬氨酸(NMDA)诱导的视网膜神经节细胞凋亡的抑制作用

目的 探讨分泌型糖蛋白Reelin对N-甲基-D-天冬氨酸(NMDA)诱导的视网膜神经节细胞(RGC)凋亡的抑制作用.方法 将大鼠RGC-5分为对照组、NMDA组、Reelin类似物组、Reelin阴性对照组和Reelin抑制物组.对照组细胞用含双抗的氨基酸双抗培养基培养,NMDA组在对照组细胞培养基中添加100μmo...     

HCN4-like immunoreactivity in rat retinal ganglion cells

Antisera directed against hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels bind to somata in the ganglion cell layer of rat and rabbit retinas, and mRNA for different HCN channel isoforms has been detected in the ganglion cell layer of mouse retina. However, previous studies neither provided evidence that any of the somata are ganglion cells (as opposed to displaced amacrine cells) nor quantified these cells. We therefore tested whether isoform-specific anti-HCN channel antisera bind to ganglion cells labeled by retrograde transport of fluorophore-coupled dextran. In flat-mounted adult rat retinas, the number of dextran-backfilled ganglion cells agreed with cell densities reported in previous studies, and anti-HCN4 antisera bound to the somata of approximately 40% of these cells. The diameter of these somata ranged from 7 to 30 microm. Consistent with localization to cell membranes, the immunoreactivity formed a thin line that circumscribed individual somata. Optic fiber layer axon fascicles, and the proximal dendrites of some ganglion cells, also displayed binding of anti-HCN4 antisera. These results suggest that the response of some mammalian retinal ganglion cells to hyperpolarization may be modulated by changes in intracellular cAMP levels, and could thus be more complex than expected from previous voltage and current recordings.