Rabbit Retinal Ganglion Cells Research Articles

An optimized method for retrograde labelling and quantification of rabbit retinal ganglion cells

Rabbits are a commonly used animal model in glaucoma research, but their application has been limited by the techniques used to assess optic nerve injury (ONI). Our study devised an optimized method for retrograde labelling and analysing rabbit retinal ganglion cells (RGCs). This method involved improvements over the conventional method regarding the stereotaxic device, the positioning of superior colliculi, the target of axonal tracer delivery, and the visualization and analysis of labelled RGCs. To evaluate its efficacy, eight New Zealand White rabbits were divided into naïve and ONI groups. Unilateral limbal buckling surgery was performed in each animal of the ONI group to induce chronic ocular hypertension (OHT). The animals of both groups were injected with indocyanine green (ICG) into the interstice between the superior colliculus and occipital lobe on each side of the brain, and their eyes were examined by confocal scanning laser ophthalmoscopy (CSLO) after 48 h. The acquired images were then analysed to quantify the number of ICG-labelled RGCs in these eyes and their loss induced by OHT. To verify the identity and changes of the labelled RGCs, the retinas of the rabbits were subjected to immunofluorescence analyses. In addition, three animals were subjected to a second ICG labelling after 12 months to determine the influence of this procedure on the long-term viability of the labelled RGCs. Our results showed that ICG-labelled RGCs were detected by CSLO throughout the retinas of all animals. These RGCs showed a distinctly higher density below the ONH and were defective in sectorial areas in OHT eyes. Their average number in the cell counting area was 3989.2 ± 414.2 and 4023.3 ± 603.4 in the right and left eyes, respectively, of the naïve animals and 2590.9 ± 1474.2 and 3966.7 ± 24.0 in the OHT and non-OHT eyes, respectively, of the ONI animals. Immunofluorescence analyses showed positive staining with Brn3a and RBPMS in the ICG-labelled RGCs and sectorial defects of the cells in the OHT eyes, similarly as observed by CSLO. The second ICG labelling after 12 months in three animals showed no appreciable changes in RGC density compared with the first one. In summary, the optimized method of rabbit RGC retrograde labelling is reliable and accurate in both naïve and ONI animals and offers an approach for longitudinal observation of RGCs in the same eyes, which suggests its potential as a powerful tool for glaucoma and optic nerve research.

Light-evoked dendritic spikes in sustained but not transient rabbit retinal ganglion cells

Dendritic computations have a central role in neuronal function, but it is unknown how cell-class heterogeneity of dendritic electrical excitability shapes physiologically engaged neuronal and circuit computations. To address this, we examined dendritic integration in closely related classes of retinal ganglion cells (GCs) using simultaneous somato-dendritic electrical recording techniques in a functionally intact circuit. Simultaneous recordings revealed sustained OFF-GCs generated powerful dendritic spikes in response to visual input that drove action potential firing. In contrast, the dendrites of transient OFF-GCs were passive and did not generate dendritic spikes. Dendritic spike generation allowed sustained, but not transient, OFF-GCs to signal into action potential output the local motion of visual stimuli to produce a continuous wave of action potential firing in adjacent cells as images moved across the retina. Conversely, this representation was highly fragmented in transient OFF-GCs. Thus, a heterogeneity of dendritic excitability defines the computations executed by classes of GCs.

Effects of tACS-Like Electrical Stimulation on Correlated Firing of Retinal Ganglion Cells: Part III.

PurposeTranscranial alternating current stimulation (tACS) is a stimulation protocol used for learning enhancement and mitigation of cognitive dysfunction. Correlated firing has been postulated to be a meta-code that links neuronal spike responses associated with a single entity and may be an important component of high-level cognitive functions. Thus, changes in the covariance firing structure of CNS neurons such as retinal ganglion cells are one potential mechanism by which tACS can exert its effects.Materials and MethodsWe used microelectrode arrays to record light-evoked spike responses of 24 retinal ganglion cells in 7 rabbit eyecup preparations and analyzed the covariance between 30 pairs of neighboring retinal ganglion cells before, during, and after 10-minute application of alternating currents of 1 microampere at 10 or 20 Hz.ResultstACS stimulation significantly changed the covariance structure of correlated firing in 60% of simultaneously recorded retinal ganglion cells. Application of tACS in the retinal preparation increased cross-covariance in 26% of cell pairs, an effect usually associated with increased light-evoked ganglion cell firing. tACS associated decreases in cross-covariance occurred in 37% of cell pairs. Increased covariance was more common in response to the first, 10-minute application of tACS in isolated retina preparation. Changes in covariance were rare after repeated stimulation, and more likely to result in decreased covariance.ConclusionRetinal ganglion cell correlated firing is modulated by 1 microampere tACS currents showing that electrical stimulation can significantly and persistently change the structure of the correlated firing of simultaneously recorded rabbit retinal ganglion cells.

토끼 방향특이성 망막 신경절세포의 니코티닉 아세틸콜린 수용체 α7과 β4 소단위의 위치 측정

토끼 방향특이성 망막 신경절세포의 니코티닉 아세틸콜린 수용체 α7과 β4 소단위의 위치 측정

Morphological identification and systematic classification of mammalian retinal ganglion cells. I. Rabbit retinal ganglion cells.

Retinal ganglion cells (RGCs) convey visual signals to 50 regions of the brain. For reasons of interest and convenience, they constitute an excellent system for the study of brain structure and function. There is general agreement that, absent a complete "parts list," understanding how the nervous system processes information will remain an elusive goal. Recent studies indicate that there are 30-50 types of ganglion cell in mouse retina, whereas only a few years ago it was still written that mice and the more visually oriented lagomorphs had less than 20 types of RGC. More than 30 years ago, I estimated that rabbits have about 40 types of RGC. The present study indicates that this number is much too low. I have employed the old but powerful method of Golgi-impregnation to rabbit retina, studying the range of component neurons in this already well-studied retinal system. Close quantitative and qualitative analyses of 1,142 RGCs in 26 retinas take into account cell body and dendritic field size, level(s) of dendritic stratification in the retina's inner plexiform layer, and details of dendritic branching. Ninety-one morphologies are recognized. Of these, at least 32 can be correlated with physiologically studied RGCs, dye-injected for morphological analysis. It is unlikely that rabbits have 91 types of RGC, but is argued here that this number lies between 60 and 70. The present study provides a "yardstick" for measuring the output of future molecular studies that may be more definitive in fixing the number of RGC types in rabbit retina.

Synaptic inputs from identified bipolar and amacrine cells to a sparsely branched ganglion cell in rabbit retina.

There are more than 30 distinct types of mammalian retinal ganglion cells, each sensitive to different features of the visual environment. In rabbit retina, they can be grouped into four classes according to their morphology and stratification of their dendrites in the inner plexiform layer (IPL). The goal of this study was to describe the synaptic inputs to one type of Class IV ganglion cell, the third member of the sparsely branched Class IV cells (SB3). One cell of this type was partially reconstructed in a retinal connectome developed using automated transmission electron microscopy (ATEM). It had slender, relatively straight dendrites that ramify in the sublamina a of the IPL. The dendrites of the SB3 cell were always postsynaptic in the IPL, supporting its identity as a ganglion cell. It received 29% of its input from bipolar cells, a value in the middle of the range for rabbit retinal ganglion cells studied previously. The SB3 cell typically received only one synapse per bipolar cell from multiple types of presumed OFF bipolar cells; reciprocal synapses from amacrine cells at the dyad synapses were infrequent. In a few instances, the bipolar cells presynaptic to the SB3 ganglion cell also provided input to an amacrine cell presynaptic to the ganglion cell. There was apparently no crossover inhibition from narrow-field ON amacrine cells. Most of the amacrine cell inputs were from axons and dendrites of GABAergic amacrine cells, likely providing inhibitory input from outside the classical receptive field.

Visual and electric spiking responses of seven types of rabbit retinal ganglion cells.

Electric stimulation of the retina via retinal implants is currently the only commercially available method to restore vision in patients suffering from a wide range of outer retinal degenerations. To improve the quality of retinal implants, it is desirable to better understand how different retinal cell classes and types respond to electric stimuli so that more effective stimulation strategies can be developed. Here, we measured the response of seven major types of retinal ganglion cells to electric stimulation. A simple series of light stimuli were used to classify cells into known types. Electric stimulation produced unique responses in almost all ganglion cell types and the electric responses typically matched elements of the corresponding light responses.

Rabbit Retinal Ganglion Cell Activation Thresholds in Response to Various Electrical Stimulation Waveforms using a Different Spatial Resolution Electrode Array

Rabbit Retinal Ganglion Cell Activation Thresholds in Response to Various Electrical Stimulation Waveforms using a Different Spatial Resolution Electrode Array

Neural architecture of the "transient" ON directionally selective (class IIb1) ganglion cells in rabbit retina, partly co-stratified with starburst amacrine cells.

Recent physiological studies coupled with intracellular staining have subdivided ON directionally selective (DS) ganglion cells of rabbit retina into two types. One exhibits more "transient" and more "brisk" responses (ON DS-t), and the other has more "sustained' and more "sluggish" responses (ON DS-s), although both represent the same three preferred directions and show preference for low stimulus velocity, as reported in previous studies of ON DS ganglion cells in rabbit retina. ON DS-s cells have the morphology of ganglion cells previously shown to project to the medial terminal nucleus (MTN) of the accessory optic system, and the MTN-projecting, class IVus1 cells have been well-characterized previously in terms of their dendritic morphology, branching pattern, and stratification. ON DS-t ganglion cells have a distinctly different morphology and exhibit heterotypic coupling to amacrine cells, including axon-bearing amacrine cells, with accompanying synchronous firing, while ON DS-s cells are not coupled. The present study shows that ON DS-t cells are morphologically identical to the previously well-characterized, "orphan" class IIb1 ganglion cell, previously regarded as a member of the "brisk-concentric" category of ganglion cells. Its branching pattern, quantitatively analyzed, is similar to that of the morphological counterparts of X and Y cells, and very different from that of the ON DS-s ganglion cell. Close analysis of the dendritic stratification of class IIb1 ganglion cells together with fiducial cells indicates that they differ from that of the ON DS-s cells. In agreement with one of the three previous studies, class IIb1/ON DS-t cells, unlike class IVus1/ON DS-s ganglion cells, in the main do not co-stratify with starburst amacrine cells. As the present study shows, however, portions of their dendrites do deviate from the main substratum, coming within range of starburst boutons. Parsimony favors DS input from starburst amacrine cells both to ON DS-s and to ON DS-t ganglion cells, given the similarity of their DS responses, but further studies will be required to substantiate the origin of the DS responses of ON DS-t cells. Previously reported OFF DS responses in ON DS-t cells, unmasked by pharmacological agents, and mediated by gap junctions with amacrine cells, suggests an unusual trans-sublaminar organization of directional selectivity in the inner plexiform layer, connecting sublamina a and sublamina b.

Identification of Synaptic Patterns of NMDA Receptor Subtypes Upon Direction-Selective Rabbit Retinal Ganglion Cells

Purpose: The objective of this study was to identify anisotropies that contribute to the directional preference of direction-selective retinal ganglion cells (DS RGCs) in the rabbit retina. We investigated the distributions of N-methyl-d-aspartate receptor 1 (NMDAR1), NMDAR2A and NMDAR2B receptor subunits in the dendritic arbors of rabbit DS RGCs.Methods: The distributions of the NMDAR subunits on the DS RGCs were determined using immunocytochemistry. DS RGCs were injected with Lucifer yellow, and the cells were identified by their characteristic morphology. The triple-labeled images of dendrites, kinesin II and NMDARs were visualized using confocal microscopy and were reconstructed from high-resolution confocal images.Results: We found no evidence of asymmetry in any of the NMDAR subunits examined on the dendritic arbors of both the ON and OFF layers of DS RGCs.Conclusions: Our results indicate that direction selectivity appears to lie in the neuronal circuitry afferent to the DS RGCs.

The Retinal Response to Sinusoidal Electrical Stimulation.

Rectangular electrical pulses are the primary stimulus waveform used in retinal prosthetics as well as many other neural stimulation applications. Unfortunately, the utility of pulsatile stimuli is limited by the inability to avoid the activation of passing axons, which can result in the distortion of the spatial patterns of elicited neural activity. Because avoiding axons would likely improve clinical outcomes, the examination of alternate stimulus waveforms is warranted. Here, we studied the response of rabbit retinal ganglion cells (RGCs) to sinusoidal electrical stimulation applied at frequencies of 5, 10, 25, and 100 Hz. Targeted RGCs were restricted to 4 common types: OFF-Brisk Transient, OFF-Sustained, ON-Brisk Transient, and ON-Sustained. Interestingly, response patterns varied between different types; the most notable difference was the relatively weak response of ON-Sustained cells to low frequencies. Calculation of total spike counts per trial revealed that lower frequencies are more charge efficient than high frequencies. Finally, experiments utilizing synaptic blockers revealed that 5 and 10 Hz activate photoreceptors while 25 and 100 Hz activate RGCs. Taken together, our results suggest that while sinusoidal electrical stimulation may provide a useful research tool, its clinical utility may be limited.

Moniliform deformation of retinal ganglion cells by formaldehyde‐based fixatives

ABSTRACTProtocols for characterizing cellular phenotypes commonly use chemical fixatives to preserve anatomical features, mechanically stabilize tissue, and stop physiological responses. Formaldehyde, diluted in either phosphate‐buffered saline or phosphate buffer, has been widely used in studies of neurons, especially in conjunction with dyes and antibodies. However, previous studies have found that these fixatives induce the formation of bead‐like varicosities in the dendrites and axons of brain and spinal cord neurons. We report here that these formaldehyde formulations can induce bead formation in the dendrites and axons of adult rat and rabbit retinal ganglion cells, and that retinal ganglion cells differ from hippocampal, cortical, cerebellar, and spinal cord neurons in that bead formation is not blocked by glutamate receptor antagonists, a voltage‐gated Na+ channel toxin, extracellular Ca2+ ion exclusion, or temperature shifts. Moreover, we describe a modification of formaldehyde‐based fixatives that prevents bead formation in retinal ganglion cells visualized by green fluorescent protein expression and by immunohistochemistry. J. Comp. Neurol. 523:545–564, 2015. © 2014 Wiley Periodicals, Inc.

Moniliform deformation of retinal ganglion cells by formaldehyde-based fixatives.

Protocols for characterizing cellular phenotypes commonly use chemical fixatives to preserve anatomical features, mechanically stabilize tissue, and stop physiological responses. Formaldehyde, diluted in either phosphate-buffered saline or phosphate buffer, has been widely used in studies of neurons, especially in conjunction with dyes and antibodies. However, previous studies have found that these fixatives induce the formation of bead-like varicosities in the dendrites and axons of brain and spinal cord neurons. We report here that these formaldehyde formulations can induce bead formation in the dendrites and axons of adult rat and rabbit retinal ganglion cells, and that retinal ganglion cells differ from hippocampal, cortical, cerebellar, and spinal cord neurons in that bead formation is not blocked by glutamate receptor antagonists, a voltage-gated Na(+) channel toxin, extracellular Ca(2+) ion exclusion, or temperature shifts. Moreover, we describe a modification of formaldehyde-based fixatives that prevents bead formation in retinal ganglion cells visualized by green fluorescent protein expression and by immunohistochemistry.

Nicotinic and muscarinic acetylcholine receptors shape ganglion cell response properties.

The purpose of this study was to evaluate the expression patterns of nicotinic and muscarinic ACh receptors (nAChRs and mAChRs, respectively) in relation to one another and to understand their effects on rabbit retinal ganglion cell response properties. Double-label immunohistochemistry revealed labeled inner-retinal cell bodies and complex patterns of nAChR and mAChR expression in the inner plexiform layer. Specifically, the expression patterns of m1, m4, and m5 muscarinic receptors overlapped with those of non-α7 and α7 nicotinic receptors in presumptive amacrine and ganglion cells. There was no apparent overlap in the expression patterns of m2 muscarinic receptors with α7 nicotinic receptors or of m3 with non-α7 nicotinic receptors. Patch-clamp recordings demonstrated cell type-specific effects of nicotinic and muscarinic receptor blockade. Muscarinic receptor blockade enhanced the center responses of brisk-sustained/G4 On and G4 Off ganglion cells, whereas nicotinic receptor blockade suppressed the center responses of G4 On-cells near the visual streak but enhanced the center responses of nonstreak G4 On-cells. Blockade of muscarinic or nicotinic receptors suppressed the center responses of brisk-sustained Off-cells and the center light responses of subsets of brisk-transient/G11 On- and Off-cells. Only nicotinic blockade affected the center responses of G10 On-cells and G5 Off-cells. These data indicate that physiologically and morphologically identified ganglion cell types have specific patterns of AChR expression. The cholinergic receptor signatures of these cells may have implications for understanding visual defects in disease states that result from decreased ACh availability.

Action potentials in retinal ganglion cells are initiated at the site of maximal curvature of the extracellular potential

Objective. The initiation of an action potential by extracellular stimulation occurs after local depolarization of the neuronal membrane above threshold. Although the technique shows remarkable clinical success, the site of action and the relevant stimulation parameters are not completely understood. Approach. Here we identify the site of action potential initiation in rabbit retinal ganglion cells (RGCs) interfaced to an array of extracellular capacitive stimulation electrodes. We determine which feature of the extracellular potential governs action potential initiation by simultaneous stimulation and recording RGCs interfaced in epiretinal configuration. Stimulation electrodes were combined to areas of different size and were presented at different positions with respect to the RGC. Main results. Based on stimulation by electrodes beneath the RGC soma and simultaneous sub-millisecond latency measurement we infer axonal initiation at the site of maximal curvature of the extracellular potential. Stimulation by electrodes at different positions along the axon reveals a nearly constant threshold current density except for a narrow region close to the cell soma. These findings are explained by the concept of the activating function modified to consider a region of lower excitability close to the cell soma. Significance. We present a framework how to estimate the site of action potential initiation and the stimulus required to cross threshold in neurons tightly interfaced to capacitive stimulation electrodes. Our results underscore the necessity of rigorous electrical characterization of the stimulation electrodes and of the interfaced neural tissue.

Direction selectivity is computed by active dendritic integration in retinal ganglion cells

Active dendritic integration is thought to enrich the computational power of central neurons. However, a direct role of active dendritic processing in the execution of defined neuronal computations in intact neural networks has not been established. Here we used multi-site electrophysiological recording techniques to demonstrate that active dendritic integration underlies the computation of direction selectivity in rabbit retinal ganglion cells. Direction-selective retinal ganglion cells fire action potentials in response to visual image movement in a preferred direction. Dendritic recordings revealed that preferred-direction moving-light stimuli led to dendritic spike generation in terminal dendrites, which were further integrated and amplified as they spread through the dendritic arbor to the axon to drive action potential output. In contrast, when light bars moved in a null direction, synaptic inhibition vetoed neuronal output by directly inhibiting terminal dendritic spike initiation. Active dendritic integration therefore underlies a physiologically engaged circuit-based computation in the retina.

The response of retinal neurons to high-frequency stimulation

Objective. High-rate pulse trains have proven to be effective in cochlear prosthetics and, more recently, have been shown to elicit a wide range of interesting response properties in axons of the peripheral nervous system. Surprisingly, the effectiveness of such trains for use in retinal prostheses has not been explored. Approach. Using cell-attached patch clamp methods, we measured the in vitro response of two rabbit retinal ganglion cell types, OFF-brisk transient (OFF-BT) and ON–OFF directionally selective (DS), to trains of biphasic pulses delivered at 2000 pulses per second (PPS). Main Results. For OFF-BT cells, response onset occurred at ∼20 µA, and maximum response occurred at ∼40 µA. Interestingly, spiking levels decreased for further increases in amplitude. In contrast, DS cells had a spiking onset at ∼25 µA and maintained strong spiking as stimulus amplitude was increased, even at the highest levels tested. Thus, a low-amplitude stimulus train at 2000 PPS (∼25 µA) will activate OFF-BT cells strongly, while simultaneously activating DS cells only weakly. In contrast, a high amplitude train (∼75 µA) will activate DS cells strongly while suppressing responses in OFF-BT cells. Significance. The response differences between cell types suggest some forms of preferential activation may be possible, and further testing is warranted. Further, the scope of the response differences found here suggests activation mechanisms that are more complex than those described in previous studies.

Visual Stimulation Reverses the Directional Preference of Direction-Selective Retinal Ganglion Cells

Direction selectivity in the retina is mediated by direction-selective ganglion cells. These cells are part of a circuit in which they are asymmetrically wired to inhibitory neurons. Thus, they respond strongly to an image moving in the preferred direction and weakly to an image moving in the opposite (null) direction. Here, we demonstrate that adaptation with short visual stimulation of a direction-selective ganglion cell using drifting gratings can reverse this cell's directional preference by 180°. This reversal is robust, long lasting, and independent of the animal's age. Our findings indicate that, even within circuits that are hardwired, the computation of direction can be altered by dynamic circuit mechanisms that are guided by visual stimulation.

A novel high electrode count spike recording array using an 81,920 pixel transimpedance amplifier-based imaging chip

Microelectrode recording arrays of 60–100 electrodes are commonly used to record neuronal biopotentials, and these have aided our understanding of brain function, development and pathology. However, higher density microelectrode recording arrays of larger area are needed to study neuronal function over broader brain regions such as in cerebral cortex or hippocampal slices. Here, we present a novel design of a high electrode count picocurrent imaging array (PIA), based on an 81,920 pixel Indigo ISC9809 readout integrated circuit camera chip. While originally developed for interfacing to infrared photodetector arrays, we have adapted the chip for neuron recording by bonding it to microwire glass resulting in an array with an inter-electrode pixel spacing of 30μm. In a high density electrode array, the ability to selectively record neural regions at high speed and with good signal to noise ratio are both functionally important. A critical feature of our PIA is that each pixel contains a dedicated low noise transimpedance amplifier (∼0.32pA rms) which allows recording high signal to noise ratio biocurrents comparable to single electrode voltage amplifier recordings. Using selective sampling of 256 pixel subarray regions, we recorded the extracellular biocurrents of rabbit retinal ganglion cell spikes at sampling rates up to 7.2kHz. Full array local electroretinogram currents could also be recorded at frame rates up to 100Hz. A PIA with a full complement of 4 readout circuits would span 1cm and could acquire simultaneous data from selected regions of 1024 electrodes at sampling rates up to 9.3kHz.

Functional expression of ionotropic glutamate receptors in the rabbit retinal ganglion cells

It has been known that retinal ganglion cells (RGCs) with distinct morphologies have different physiological properties. It was hypothesized that different functions of RGCs may in part result from various expressions of N-methyl- d-aspartate (NMDA), α-amino-3-hydroxyl-5-methyl-isoxazole-4-propinoic acid (AMPA), and kainic acid (KA) receptors on their dendrites. In the present study, we aimed to characterize the functional expression of AMPA and NMDA receptors of morphologically identified RGCs in the wholemount rabbit retina. The agmatine (AGB) activation assay was used to reveal functional expression of ionotropic glutamate receptors after the RGCs were targeted by injecting Neurobiotin. To examine the excitability of these glutamate receptors in an agonist specific manner, the lower concentrations of AMPA (2 μM) and NMDA (100 μM) were chosen to examine G7 (ON–OFF direction selective ganglion cells) and G11 (alpha ganglion cells) types of RGCs. We found that less than 40% of G7 type RGCs had salient AGB activation when incubated with 2 μM AMPA or 100 μM NMDA. The G11 type RGCs also showed similar activation frequencies, except that all of the OFF subtype examined had no AGB permeation under the same AMPA concentration. These results suggest that RGCs with large somata (G7 and G11 types) may express various heterogeneous functional ionotropic glutamate receptors, thus in part rendering their functional diversity.