Digital reconstruction, quantitative morphometric analysis, and membrane properties of bipolar cells in the rat retina.

A basic principle of neuroscience is that structure reflects function. This has led to numerous attempts to characterize the complete morphology of types of neurons throughout the central nervous system. The ability to acquire and analyze complete neuronal morphologies has advanced with continuous technological developments for over 150 years, with progressive refinements and increased understanding of the precise anatomical details of different types of neurons. Bipolar cells of the mammalian retina are short-range projections neurons that link the outer and inner retina. Their dendrites contact and receive input from the terminals of the light-sensing photoreceptors in the outer plexiform layer and their axons descend through the inner nuclear and inner plexiform layers to stratify at different levels of the inner plexiform layer. The stratification level of the axon terminals of different types of bipolar cells in the inner plexiform layer determines their synaptic connectivity and is an important basis for the morphological classification of these cells. Between 10 and 15 different types of cone bipolar cells have been identified in different species and they can be divided into ON-cone bipolar cells (that depolarize to the onset of light) and OFF-cone bipolar cells (that depolarize to the offset of light). Different types of cone bipolar cells are thought to be responsible for coding and transmitting different features of our visual environment and generating parallel channels that uniquely filter and transform the inputs from the photoreceptors. There is a lack of detailed morphological data for bipolar cells, especially for the rat, where biophysical mechanisms have been most extensively studied. The work presented in this thesis provides the groundwork for the future goal of developing morphologically realistic compartmental models for cone and rod bipolar cells. First, the contribution of gap junctions to the membrane properties, specifically input resistance, of bipolar cells was investigated. Gap junctions are ubiquitous within the retina, but it remains to be determined whether the strength of coupling between specific cell types is sufficiently strong for the cells to be functionally coupled via electrical synapses. There are gap junctions between the same class of bipolar cells, and this appears to be a common circuit motif in the vertebrate retina. Surprisingly, our results suggested that the gap junctions between OFF-cone bipolar cells do not support consequential electrical coupling. This provides an important first step both to elucidate the potential roles for these gap junctions, and also for the development of compartmental models for cone bipolar cells. Second, from image stacks acquired from multiphoton excitation microscopy, quantitative morphological reconstructions and detailed morphological analysis were performed with fluorescent dye-filled cone and rod bipolar cells. Compared to previous descriptions, the extent and complexity of branching of the axon terminals was surprisingly high. By precisely quantifying the level of stratification of the axon terminals in the inner plexiform layer, we have generated a reference system for the reliable classification of individual cells in future studies that are focused on correlating physiological and morphological properties. The workflow that we have implemented can be readily extended to the development of morphologically realistic compartmental models for these neurons.Doktorgradsavhandlin

The Role of Non-Linearities in Visual Perception studied with a Computational Model of the Vertebrate Retina

Processing of visual stimuli in the vertebrate retina is complex and diverse. The retinal output to the higher centres of the nervous system, mediated by ganglion cells, consists of several different channels. Neurons in these channels can have very distinct response properties, which originate in different retinal pathways. In this work, the retinal origins and possible functional implications of the segregation of visual pathways will be investigated with a detailed, biologically realistic computational model of the retina. This investigation will focus on the two main retino-cortical pathways in the mammalian retina, the parvocellular and magnocellular systems, which are crucial for conscious visual perception. These pathways differ in two important aspects. The parvocellular system has a high spatial, but low temporal resolution. Conversely, the magnocellular system has a high temporal fidelity, spatial sampling however is less dense than for parvocellular cells. Additionally, the responses of magnocellular ganglion cells can show pronounced nonlinearities, while the parvocellular system is essentially linear. The origin of magnocellular nonlinearities is unknown and will be investigated in the first part of this work. As their main source, the results suggest specific properties of the photoreceptor response and a specialised amacrine cell circuit in the inner retina. The results further show that their effect combines in a multiplicative way. The model is then used to examine the influence of nonlinearities on the responses of ganglion cells in the presence of involuntary fixational eye movements. Two different stimulus conditions will be considered: visual hyperacuity and motion induced illusions. In both cases, it is possible to directly compare properties of the ganglion cell population response with psychophysical data, which allows for an analysis of the influence of different components of the retinal circuitry. The simulation results suggest an important role for nonlinearities in the magnocellular stream for visual perception in both cases. First, it will be shown how nonlinearities, triggered by fixational eye movements, can strongly enhance the spatial precision of magnocellular ganglion cells. As a result, their performance in a hyperacuity task can be equal to or even surpass that of the parvocellular system. Second, the simulations imply that the origin of some of the illusory percepts elicited by fixational eye movements could be traced back to the nonlinear properties of magnocellular ganglion cells. As these activity patterns strongly differ from those in the parvocellular system, it appears that the magnocellular system can strongly dominate visual perception in certain conditions. Taken together, the results of this theoretical study suggest that retinal nonlinearities may be important for and strongly influence visual perception. The model makes several experimentally verifiable predictions to further test and quantify these findings. Furthermore, models investigating higher visual processing stages may benefit from this work, which could provide the basis to produce realistic afferent input

Divergence of visual channels in the inner retina

Bipolar cells form parallel channels that carry visual signals from the outer to the inner retina. Each type of bipolar cell is thought to carry a distinct visual message to select types of amacrine cells and ganglion cells. However, the number of ganglion cell types exceeds that of the bipolar cells providing their input, suggesting that bipolar cell signals diversify on transmission to ganglion cells. We explored in the salamander retina how signals from individual bipolar cells feed into multiple ganglion cells and found that each bipolar cell was able to evoke distinct responses among ganglion cells, differing in kinetics, adaptation and rectification properties. This signal divergence resulted primarily from interactions with amacrine cells that allowed each bipolar cell to send distinct signals to its target ganglion cells. Our findings indicate that individual bipolar cell–ganglion cell connections have distinct transfer functions. This expands the number of visual channels in the inner retina and enhances the computational power and feature selectivity of early visual processing

Glycine receptor subunits -a2 and a3 participate in different inhibitory circuits that alter the receptive field organization of on- and off-center retinal ganglion cells.

In the retina, the receptive fields (RFs) of most neurons are comprised of an excitatory center and a suppressive surround. Retinal ganglion cell (RGC) RF center excitatory input arises from bipolar cell (BC) inputs, while their surround arises from lateral inhibitory inputs. Because of the availability of selective antagonists the role of GABAergic inputs has been well defined. In contrast, the role of individual glycine receptor (GlyR) subunit inhibition is less clear because the antagonist, strychnine, blocks all GlyR subunit combinations. To define individual retinal circuits that utilize specific glycinergic subunits, I examined maintained and visually-evoked responses of ON- and OFF-center GCs from mice lacking expression of the GlyRa2 (Glra2-/-) or GlyRa3 (Glra3-/-) subunits to those of C57Bl/6J (WT) RGCs using an in vivo extracellular approach. Previous observations have defined glycine and GABA inputs across BC classes and in a variety of amacrine and RGCs. Using this information and by comparing the responses of WT vs. Glra2-/- and Glra3-/- RGCs; I conclude that both subunits modulate local RF interactions. Within the On pathway, GlyRa2 and GlyRa3 inputs play similar roles. Their responses predict that they participate in serial inhibitory circuits that decrease a direct GABAergic inhibition that modulates maintained, but not peak firing rates. In contrast within the Off pathway, GlyRa2 and GlyRa3 inputs define two populations of RGCs. In one, GlyRa2 participates in a serial inhibitory circuit that modulates maintained firing, whereas in the other, GlyRa,3 mediates direct inhibition that controls the peak firing rate. Only GlyRa2 modulates lateral interactions to the RF surround where it mediates a direct inhibitory input to all OFF-center RGCs. My results suggest that GlyRa2 and GlyRa3 inputs define two populations of OFF-center RGCs. In addition, both subunits participate in retinal circuits that can be distinguished not only by the RGC RF center type, but also by the type of inhibitory circuit. These results are the first demonstration of subunit specific control of RGC visual responses and, are the first evidence of serial glycine to GABA as well as glycine to glycine circuits in the retina

Glycine receptor subunits -a2 and a3 participate in different inhibitory circuits that alter the receptive field organization of on- and off-center retinal ganglion cells.

In the retina, the receptive fields (RFs) of most neurons are comprised of an excitatory center and a suppressive surround. Retinal ganglion cell (RGC) RF center excitatory input arises from bipolar cell (BC) inputs, while their surround arises from lateral inhibitory inputs. Because of the availability of selective antagonists the role of GABAergic inputs has been well defined. In contrast, the role of individual glycine receptor (GlyR) subunit inhibition is less clear because the antagonist, strychnine, blocks all GlyR subunit combinations. To define individual retinal circuits that utilize specific glycinergic subunits, I examined maintained and visually-evoked responses of ON- and OFF-center GCs from mice lacking expression of the GlyRa2 (Glra2-/-) or GlyRa3 (Glra3-/-) subunits to those of C57Bl/6J (WT) RGCs using an in vivo extracellular approach. Previous observations have defined glycine and GABA inputs across BC classes and in a variety of amacrine and RGCs. Using this information and by comparing the responses of WT vs. Glra2-/- and Glra3-/- RGCs; I conclude that both subunits modulate local RF interactions. Within the On pathway, GlyRa2 and GlyRa3 inputs play similar roles. Their responses predict that they participate in serial inhibitory circuits that decrease a direct GABAergic inhibition that modulates maintained, but not peak firing rates. In contrast within the Off pathway, GlyRa2 and GlyRa3 inputs define two populations of RGCs. In one, GlyRa2 participates in a serial inhibitory circuit that modulates maintained firing, whereas in the other, GlyRa,3 mediates direct inhibition that controls the peak firing rate. Only GlyRa2 modulates lateral interactions to the RF surround where it mediates a direct inhibitory input to all OFF-center RGCs. My results suggest that GlyRa2 and GlyRa3 inputs define two populations of OFF-center RGCs. In addition, both subunits participate in retinal circuits that can be distinguished not only by the RGC RF center type, but also by the type of inhibitory circuit. These results are the first demonstration of subunit specific control of RGC visual responses and, are the first evidence of serial glycine to GABA as well as glycine to glycine circuits in the retina

Extracellular electrical stimulation of retinal ganglion cells

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1994.Includes bibliographical references (p. 106-110).by Andrew Eli Grumet.M.S

Doctor of Philosophy

dissertationIt is imperative to obtain a complete network graph of at least one representative retina if we are to fully understand vertebrate vision. Synaptic connectomics endeavors to construct such graphs. Though previously prevented by hardware and software limitations, the creation of customized viewing and analysis software, affordable data storage, and advances in electron imaging platform control now permit connectome assembly and analysis. The optimal strategy for building complete connectomes utilizes automated transmission electron imaging with 2 nm or better resolution, molecular tags for cell identification, open access data volumes for navigation, and annotation with open source tools to build three-dimensional cell libraries, complete network diagrams, and connectivity databases. In a few years, the first retinal connectome analyses reveal that many well-studied cells participate in much richer networks than expected. Collectively, these results impel a refactoring of the inner plexiform layer, while providing proof of concept for connectomics as a game-changing approach for a new era of scientific discovery

Understanding object motion encoding in the mammalian retina.

Phototransduction, transmission of visual information down the optic nerve incurs delays on the order of 50 – 100ms. This implies that the neuronal representation of a moving object should lag behind the object’s actual position. However, studies have demonstrated that the visual system compensates for neuronal delays using a predictive mechanism called phase advancing, which shifts the population response toward the leading edge of a moving object’s retinal image. To understand how this compensation is achieved in the retina, I investigated cellular and synaptic mechanisms that drive phase advancing. I used three approaches, each testing phase advancing at a different organizational level within the mouse retina. First, I studied phase advancing at the level of ganglion cell populations, using two-photon imaging of visually evoked calcium responses. I found populations of phase advancing OFF-type, ON-type, ON-OFF type, and horizontally tuned directionally selective ganglion cells. Second, I measured synaptic current responses of individual ganglion cells with patch-clamp electrophysiology, and I used a computational model to compare the observed responses to simulated responses based on the ganglion cell’s spatio-temporal receptive fields. Third, I tested whether phase advancing originates presynaptic to ganglion cells, by assessing phase advancing at the level of bipolar cell glutamate release using two-photon imaging of the glutamate biosensor iGluSnFR expressed in the inner plexiform layer. Based on the results of my experiments, I conclude that bipolar and ganglion cell receptive field structure generates phase advanced responses and acts to compensate for neuronal delays within the retina

Transient receptor potential cation channel, subfamilies V, member 1 (TRPV1) and M, member 1 (TRPM1) contribute to neural signaling in mouse retina.

The retina processes light information through parallel pathways in order to extract and encode the visual scene. Light information is transmitted to the brain through approximately 30 ganglion cells (GCs), the retinal output neurons. Trp channels modulate the responses of retinal neurons within specific pathways. The study of the expression and function of the majority of Trp channels in the retina is largely in its infancy. My dissertation first investigated the expression and function of the transient receptor potential vanilloid-1 (TRPV1) receptor/channel in the retina. TRPV1, the first cloned and most highly studied Trp channel in the peripheral nervous system, is a non-selective cation channel with an affinity for Ca2+. The channel can be activated by capsaicin, acid, endovanilloids, noxious heat or pressure (Moreira et al., 2012). Located on the peripheral and central terminals of nociceptive fibers in the PNS and in limited areas of the CNS (Cavanaugh et al, 2011b). TRPV1 plays a role in inflammation, chronic pain, nociceptor sensitization and desensitization, long-term depression and potentiation, and apoptosis. The role of TRPV1 in the retina is not known. Using the electroretinogram (ERG), a mass potential that assesses the function of photoreceptors and bipolar cells, the TRPV1 knockout mouse appears normal. However, TRPV1 is thought to play a role in calcium regulation and glaucoma (Sappington et al., 2009 & Leonelli et al., 2010) so we investigated its role in normal visual transduction in the inner retina. To investigate TRPV1 modulation, I recorded GC spiking responses to light stimuli from mice which either express or lack TRPV1 protein. I found that TRPV1 is critical for: 1. GC responses to dim light. 2. Sustained responses to light 3. Surround suppression of GCs to large spots. Further, I investigate the specific retinal cells that express TRPV1. I used TRPV1cre mice with genetic or viral methods to fluorescently label neurons that express TRPV1. I determined TRPV1 is expressed in four classes of amacrine and three classes of ganglion cells in the inner retina. My results indicate TRPV1 activity in the amacrine cells enhances the sustained spiking responses in GCs. In this way, TRPV1 likely enhances the perception of subtle details in the visual world. TRPV1 also is expressed in subsets of intrinsically photosensitive GCs, which are known to play a role in circadian photoentrainment. TRPV1 therefore has the potential to modulate circadian photoentrainment or other non-image forming visual functions as well. The role of TRPM1 in the retina is well known. It is required for signaling through the ON pathway, which detects light increments. Responses through the vii ON pathway are initiated by synapses between rod and cone photoreceptors with ON bipolar cells (BCs). The human disease, complete congenital stationary night blindness (cCSNB) results from a disruption in signaling within the ON BC mGluR6 G-protein coupled cascade, which culminates in the opening of the TRPM1 channel and signaling through ON BCs. I helped expand our understanding of the role of TRPM1 in the retina by investigating the expression and function of leucine rich repeat immunoglobulin like transmembrane protein 3 (LRIT3), a novel protein component in the mGluR6-TRPM1 signalplex that was found mutated within cCSNB patients and a knockout mouse (Zeitz et al., 2013; Neuillé et al., 2014). The function of LRIT3 within the cascade remains unknown. To better understand the role of LRIT3, we examined retinal structure and function. We compared the structure of the pre and postsynaptic elements in the OPL of WT and Lrit3-/- mice using a variety of antibodies and with confocal microscopy. We assessed overall retinal function with ERG and GC spontaneous and visually evoked activity with single cell and multielectrode array recordings. The overall laminar structure of the Lrit3-/- retina is similar to WT. Consistent with published results and other cCSNB mouse models, Lrit3-/- mouse dark- and lightadapted ERGs have a normal a-wave, but lack a b-wave. The dendritic terminals of Lrit3-/- ON BCs lack expression of nyctalopin and TRPM1. Lrit3-/- mice significantly differ from other cCSNB mutants. Cone ON BCs lack expression of mGluR6, GPR179 and RGS11, whereas rod BCs maintain expression of these proteins. LRIT3 is necessary for expression and localization of nyctalopin and TRPM1 to the ON BC dendrites. As expected there are no ON responses, but surprisingly very few (~22%) Lrit3-/- GCs have even OFF responses. Lrit3-/- OFF BCs express functional kainate glutamate receptors. However, Lrit3-/- OFF BC and OFF GCs have significantly smaller response to light decrements than WT. Like all other mouse models of cCSNB, LRIT3 is critical to signaling in ON BCs, however, unlike all other cCSNB models, LRIT3 also has a trans-synaptic role in enhancing glutamate transmission from cones to BCs

An Optoelectronic Stimulator for Retinal Prosthesis

Retinal prostheses require the presence of viable population of cells in the inner retina. Evaluations of retina with Age-Related Macular Degeneration (AMD) and Retinitis Pigmentosa (RP) have shown a large number of cells remain in the inner retina compared with the outer retina. Therefore, vision loss caused by AMD and RP is potentially treatable with retinal prostheses. Photostimulation based retinal prostheses have shown many advantages compared with retinal implants. In contrary to electrode based stimulation, light does not require mechanical contact. Therefore, the system can be completely external and not does have the power and degradation problems of implanted devices. In addition, the stimulating point is flexible and does not require a prior decision on the stimulation location. Furthermore, a beam of light can be projected on tissue with both temporal and spatial precision. This thesis aims at fi nding a feasible solution to such a system. Firstly, a prototype of an optoelectronic stimulator was proposed and implemented by using the Xilinx Virtex-4 FPGA evaluation board. The platform was used to demonstrate the possibility of photostimulation of the photosensitized neurons. Meanwhile, with the aim of developing a portable retinal prosthesis, a system on chip (SoC) architecture was proposed and a wide tuning range sinusoidal voltage-controlled oscillator (VCO) which is the pivotal component of the system was designed. The VCO is based on a new designed Complementary Metal Oxide Semiconductor (CMOS) Operational Transconductance Ampli er (OTA) which achieves a good linearity over a wide tuning range. Both the OTA and the VCO were fabricated in the AMS 0.35 µm CMOS process. Finally a 9X9 CMOS image sensor with spiking pixels was designed. Each pixel acts as an independent oscillator whose frequency is controlled by the incident light intensity. The sensor was fabricated in the AMS 0.35 µm CMOS Opto Process. Experimental validation and measured results are provided