Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase^1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina^5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation⁶. In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.Download video file.^{(32M, mov)}     

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp ^{1, 2, 3} and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell''s instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.     

视网膜中的自主感光神经节细胞

视网膜中少数神经节细胞能够合成感光蛋白--黑视素(melanopsin),因此具备了自主感光的能力,被称为自主感光神经节细胞(intrinsically photosensitive retinal ganglion cells,ipRGCs).ipRGCs可根据树突形态和分层位置的差异分为五个不同的亚型,其轴突主要投...     

Lactate-Mediated Protection of Retinal Ganglion Cells

Loss of retinal ganglion cells (RGCs) is a leading cause of blinding conditions. The purpose of this study was to evaluate the effect of extracellular l-lactate on RGC survival facilitated through lactate metabolism and ATP production. We identified lactate as a preferred energy substrate over glucose in murine RGCs and showed that lactate metabolism and consequently increased ATP production are crucial components in promoting RGC survival during energetic crisis. Lactate was released to the extracellular environment in the presence of glucose and detained intracellularly during glucose deprivation. Lactate uptake and metabolism was unaltered in the presence and absence of glucose. However, the ATP production declined significantly for 24 h of glucose deprivation and increased significantly in the presence of lactate. Finally, lactate exposure for 2 and 24 h resulted in increased RGC survival during glucose deprivation. In conclusion, the metabolic pathway of lactate in RGCs may be of great future interest to unravel potential pharmaceutical targets, ultimately leading to novel therapies in the prevention of blinding neurodegenerative diseases, for example, glaucoma.     

Protection of Retinal Ganglion Cells and Retinal Vasculature by Lycium Barbarum Polysaccharides in a Mouse Model of Acute Ocular Hypertension

Acute ocular hypertension (AOH) is a condition found in acute glaucoma. The purpose of this study is to investigate the protective effect of Lycium barbarum polysaccharides (LBP) and its protective mechanisms in the AOH insult. LBP has been shown to exhibit neuroprotective effect in the chronic ocular hypertension (COH) experiments. AOH mouse model was induced in unilateral eye for one hour by introducing 90 mmHg ocular pressure. The animal was fed with LBP solution (1 mg/kg) or vehicle daily from 7 days before the AOH insult till sacrifice at either day 4 or day 7 post insult. The neuroprotective effects of LBP on retinal ganglion cells (RGCs) and blood-retinal-barrier (BRB) were evaluated. In control AOH retina, loss of RGCs, thinning of IRL thickness, increased IgG leakage, broken tight junctions, and decreased density of retinal blood vessels were observed. However, in LBP-treated AOH retina, there was less loss of RGCs with thinning of IRL thickness, IgG leakage, more continued structure of tight junctions associated with higher level of occludin protein and the recovery of the blood vessel density when compared with vehicle-treated AOH retina. Moreover, we found that LBP provides neuroprotection by down-regulating RAGE, ET-1, Aβ and AGE in the retina, as well as their related signaling pathways, which was related to inhibiting vascular damages and the neuronal degeneration in AOH insults. The present study suggests that LBP could prevent damage to RGCs from AOH-induced ischemic injury; furthermore, through its effects on blood vessel protection, LBP would also be a potential treatment for vascular-related retinopathy.     

鱼类三叉神经节的形态及神经节细胞的分布

目的:观察三叉神经节的形态结构及神经节细胞的分布。方法:用罗非鱼,进行40g/L甲醛灌注固定,观察三叉神经节的位置及分支分布,取三叉神经节,根及分支进行连续切片制作三维立体图像,观察神经节细胞的分布。结果:①三叉神经根在菱脑高度进出脑。②三叉神经节位于眼眶与颅腔之间的骨组织中。③从神经节发出的第一支(眼神经)通过眼眶的背侧分布于吻侧部,第二支(上颌神经)通过眼眶的腹侧分布于上颌部,第三支(下颌神经)通过眼眶的最腹侧分布于下颌部。④神经节细胞在神经节内背腹方向排列的一群细胞团。结论:罗非鱼三叉神经节是独立存在的,与其它鱼类的神经节有明显差异。     

Intrinsic Photosensitive Retinal Ganglion Cells in the Diurnal Rodent,Arvicanthis ansorgei

Intrinsically photosensitive retinal ganglion cells (ipRGCs) represent a new class of photoreceptors which support a variety of non-image forming physiological functions, such as circadian photoentrainment, pupillary light reflex and masking responses to light. In view of the recently proposed role of retinal inputs for the regulation of diurnal and nocturnal behavior, we performed the first deep analysis of the ipRGC system in a diurnal rodent model, Arvicanthisansorgei, and compared the anatomical and physiological properties of ipRGCs with those of nocturnal mice. Based on somata location, stratification pattern and melanopsin expression, we identified two main ipRGC types in the retina of Arvicanthis: M1, constituting 74% of all ipRGCs and non-M1 (consisting mainly of the M2 type) constituting the following 25%. The displaced ipRGCs were rarely encountered. Phenotypical staining patterns of ganglion cell markers showed a preferential expression of Brn3 and neurofilaments in non-M1 ipRGCs. In general, the anatomical properties and molecular phenotyping of ipRGCs in Arvicanthis resemble ipRGCs of the mouse retina, however the percentage of M1 cells is considerably higher in the diurnal animal. Multi-electrode array recordings (MEA) identified in newborn retinas of Arvicanthis three response types of ipRGCs (type I, II and III) which are distinguished by their light sensitivity, response strength, latency and duration. Type I ipRGCs exhibited a high sensitivity to short light flashes and showed, contrary to mouse type I ipRGCs, robust light responses to 10 ms flashes. The morphological, molecular and physiological analysis reveals very few differences between mouse and Arvicanthis ipRGCs. These data imply that the influence of retinal inputs in defining the temporal niche could be related to a stronger cone input into ipRGCs in the cone-rich Arvicanthis retina, and to the higher sensitivity of type I ipRGCs and elevated proportion of M1 cells.     

Retinal Ganglion Cells are Resistant to Photoreceptor Loss in Retinal Degeneration

The rapid and massive degeneration of photoreceptors in retinal degeneration might have a dramatic negative effect on retinal circuits downstream of photoreceptors. However, the impact of photoreceptor loss on the morphology and function of retinal ganglion cells (RGCs) is not fully understood, precluding the rational design of therapeutic interventions that can reverse the progressive loss of retinal function. The present study investigated the morphological changes in several identified RGCs in the retinal degeneration rd1 mouse model of retinitis pigmentosa (RP), using a combination of viral transfection, microinjection of neurobiotin and confocal microscopy. Individual RGCs were visualized with a high degree of detail using an adeno-associated virus (AAV) vector carrying the gene for enhanced green fluorescent protein (EGFP), allowed for large-scale surveys of the morphology of RGCs over a wide age range. Interestingly, we found that the RGCs of nine different types we encountered were especially resistant to photoreceptor degeneration, and retained their fine dendritic geometry well beyond the complete death of photoreceptors. In addition, the RGC-specific markers revealed a remarkable degree of stability in both morphology and numbers of two identified types of RGCs for up to 18 months of age. Collectively, our data suggest that ganglion cells, the only output cells of the retina, are well preserved morphologically, indicating the ganglion cell population might be an attractive target for treating vision loss.     

Directional Summation in Non-direction Selective Retinal Ganglion Cells

Retinal ganglion cells receive inputs from multiple bipolar cells which must be integrated before a decision to fire is made. Theoretical studies have provided clues about how this integration is accomplished but have not directly determined the rules regulating summation of closely timed inputs along single or multiple dendrites. Here we have examined dendritic summation of multiple inputs along On ganglion cell dendrites in whole mount rat retina. We activated inputs at targeted locations by uncaging glutamate sequentially to generate apparent motion along On ganglion cell dendrites in whole mount retina. Summation was directional and dependent13 on input sequence. Input moving away from the soma (centrifugal) resulted in supralinear summation, while activation sequences moving toward the soma (centripetal) were linear. Enhanced summation for centrifugal activation was robust as it was also observed in cultured retinal ganglion cells. This directional summation was dependent on hyperpolarization activated cyclic nucleotide-gated (HCN) channels as blockade with ZD7288 eliminated directionality. A computational model confirms that activation of HCN channels can override a preference for centripetal summation expected from cell anatomy. This type of direction selectivity could play a role in coding movement similar to the axial selectivity seen in locust ganglion cells which detect looming stimuli. More generally, these results suggest that non-directional retinal ganglion cells can discriminate between input sequences independent of the retina network.     

Expression of Aquaporin-6 in Rat Retinal Ganglion Cells

Several aquaporins (AQPs) have been identified to be present in the eyes, and it has been suggested that they are involved in the movement of water and small solutes. AQP6, which has low water permeability and transports mainly anions, was recently discovered in the eyes. In the present study, we investigate the localization of AQP6 in the rat retina and show that AQP6 is selectively localized to the ganglion cell layer and the outer plexiform layer. Along with the gradual decrease in retinal ganglion cells after a crushing injury of optic nerve, immunofluorescence signals of AQP6 gradually decreased. Confocal microscope images confirmed AQP6 expression in retinal ganglion cells and Müller cells in vitro. Therefore, AQP6 might participate in water and anion transport in these cells.     

Calcium-Dependent Increases in Protein Kinase-A Activity in Mouse Retinal Ganglion Cells Are Mediated by Multiple Adenylate Cyclases

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.     

小鼠慢性高眼压模型下视网膜神经节细胞的损伤

目的:通过巩膜外静脉烧烙术建立慢性高眼压模型,研究小鼠慢性高眼压状态下视网膜神经节细胞的凋亡情况.方法:取C57BL/6J小鼠30只.3只作为空白对照组,其余27只右眼为实验眼,左眼为对照眼.术前用iCare眼压计测量眼压,按巩膜外静脉烧烙法建立慢性高眼压模型,术后用iCare眼压计每日监测眼压.分剐取空白对照组6眼,术后1w、4 w造模成功的小鼠各8只16眼眼球,石蜡切片行Tunel法,荧光显微镜下采集图像.小鼠眼压的组间比较采用t检验.结果:给予巩膜外静脉烧烙术后1d、1w、4w小鼠慢性高眼压眼眼压(11.15±0.98、10.65±0.95、10.35±1.05)与对照眼(6.40±0.95、6.35±1.05、6.50±1.15)相比,差异有统计学意义(t=10.77～18.08,P＜0.001).Tunel法结果显示,正常小鼠空白对照组未见明显凋亡的视网膜神经节细胞.慢性高眼压组术后1w、4w可见Tunel阳性表达.而对照组术后1w及4w均未见Tunel阳性表达.结论:巩膜外静脉烧灼法能诱导出持续的肯定的小鼠慢性高眼压模型,慢性高眼压状态下小鼠视网膜神经节细胞发生凋亡,细胞凋亡是小鼠慢性高眼压状态下视网膜神经节细胞损伤的主要方式.     

青光眼视网膜神经节细胞凋亡的研究进展

青光眼视神经损伤的最后共同通路为视网膜神经节细胞的凋亡。但确切机制尚未阐明。为此,人们进行了大量相关体内、体外实验并取得一定成果。本文从凋亡的激发因素、信号传导及基因调控加以阐述。     

Treatment In vitro of Retinal Cells with IL-4 Increases the Survival of Retinal Ganglion Cells: The Involvement of BDNF

Interleukin 4 (IL-4) is a pleiotropic cytokine involved in many functions during the development as well as in adult life. Previous work from our group demonstrated, in vitro, that this interleukin is able to prevent rat retinal ganglion cells death after axotomy. The aim of the present study was to investigate the signaling pathways involved in this trophic effect, particularly the cAMP pathway and also to demonstrate the expression of IL-4 in retinas at different stages of post natal development. Our results show that the trophic effect of IL-4 on rat retinal ganglion cells is dependent on the activation of Janus Kinase 3, Protein Kinase A, c-Jun N-terminal Kinase and Tropomyosin related Kinase receptors, on the increase in intracellular calcium levels, on polypeptide release and on the endogenous Brain Derived Neurotrophic Factor (BDNF). We also observed that treatment with IL-4 enhances c-AMP response element binding and Mitogen Activated Protein Kinase phosphorylation and increases the expression of BDNF. Concerning the IL-4 expression our data show an increase in IL-4 levels during post natal development. Taken together our results demonstrate that the trophic effect of IL-4 on retinal ganglion cells of newborn rats is mediated by cAMP pathway and BDNF release.     

Spatial Relationships between GABAergic and Glutamatergic Synapses on the Dendrites of Distinct Types of Mouse Retinal Ganglion Cells across Development

Neuronal output requires a concerted balance between excitatory and inhibitory (I/E) input. Like other circuits, inhibitory synaptogenesis in the retina precedes excitatory synaptogenesis. How then do neurons attain their mature balance of I/E ratios despite temporal offset in synaptogenesis? To directly compare the development of glutamatergic and GABAergic synapses onto the same cell, we biolistically transfected retinal ganglion cells (RGCs) with PSD95CFP, a marker of glutamatergic postsynaptic sites, in transgenic Thy1­YFPγ2 mice in which GABA_A receptors are fluorescently tagged. We mapped YFPγ2 and PSD95CFP puncta distributions on three RGC types at postnatal day P12, shortly before eye opening, and at P21 when robust light responses in RGCs are present. The mature I_GABA/E ratios varied among ON-Sustained (S) A-type, OFF-S A-type, and bistratified direction selective (DS) RGCs. These ratios were attained at different rates, before eye-opening for ON-S and OFF-S A-type, and after eye-opening for DS RGCs. At both ages examined, the I_GABA/E ratio was uniform across the arbors of the three RGC types. Furthermore, measurements of the distances between neighboring PSD95CFP and YFPγ2 puncta on RGC dendrites indicate that their local relationship is established early in development, and cannot be predicted by random organization. These close spatial associations between glutamatergic and GABAergic postsynaptic sites appear to represent local synaptic arrangements revealed by correlative light and EM reconstructions of a single RGC''s dendrites. Thus, although RGC types have different I_GABA/E ratios and establish these ratios at separate rates, the local relationship between excitatory and inhibitory inputs appear similarly constrained across the RGC types studied.     

Network Oscillations Drive Correlated Spiking of ON and OFF Ganglion Cells in the rd1 Mouse Model of Retinal Degeneration

Following photoreceptor degeneration, ON and OFF retinal ganglion cells (RGCs) in the rd-1/rd-1 mouse receive rhythmic synaptic input that elicits bursts of action potentials at ∼10 Hz. To characterize the properties of this activity, RGCs were targeted for paired recording and morphological classification as either ON alpha, OFF alpha or non-alpha RGCs using two-photon imaging. Identified cell types exhibited rhythmic spike activity. Cross-correlation of spike trains recorded simultaneously from pairs of RGCs revealed that activity was correlated more strongly between alpha RGCs than between alpha and non-alpha cell pairs. Bursts of action potentials in alpha RGC pairs of the same type, i.e. two ON or two OFF cells, were in phase, while bursts in dissimilar alpha cell types, i.e. an ON and an OFF RGC, were 180 degrees out of phase. This result is consistent with RGC activity being driven by an input that provides correlated excitation to ON cells and inhibition to OFF cells. A2 amacrine cells were investigated as a candidate cellular mechanism and found to display 10 Hz oscillations in membrane voltage and current that persisted in the presence of antagonists of fast synaptic transmission and were eliminated by tetrodotoxin. Results support the conclusion that the rhythmic RGC activity originates in a presynaptic network of electrically coupled cells including A2s via a Na⁺-channel dependent mechanism. Network activity drives out of phase oscillations in ON and OFF cone bipolar cells, entraining similar frequency fluctuations in RGC spike activity over an area of retina that migrates with changes in the spatial locus of the cellular oscillator.