Retinal Circuitry Research Articles

The influences of metabotropic receptor activation on cellular signaling and synaptic function in amacrine cells

Amacrine cells receive glutamatergic input from bipolar cells and GABAergic, glycinergic, cholinergic, and dopaminergic input from other amacrine cells. Glutamate, GABA, glycine, and acetylcholine (ACh) interact with ionotropic receptors and it is these interactions that form much of the functional circuitry in the inner retina. However, glutamate, GABA, ACh, and dopamine also activate metabotropic receptors linked to second messenger pathways that have the potential to modify the function of individual cells as well as retinal circuitry. Here, the physiological effects of activating dopamine receptors, metabotropic glutamate receptors, GABAB receptors, and muscarinic ACh receptors on amacrine cells will be discussed. The retina also expresses metabotropic receptors and the biochemical machinery associated with the synthesis and degradation of endocannabinoids and sphingosine-1-phosphate (S1P). The effects of activating cannabinoid receptors and S1P receptors on amacrine cell function will also be addressed.

Shedding light on class-specific wiring: development of intrinsically photosensitive retinal ganglion cell circuitry.

Neural circuits associated with retinal ganglion cells have long been used as models for investigating the mechanisms that govern circuit development and function. Similar to neurons in the brain, retinal ganglion cells are subdivided into distinct classes based upon their morphology, physiology, and patterns of connectivity. Newly developed transgenic tools in which individual classes of retinal ganglion cells are labeled with reporter proteins have recently provided a method to study the development of their class-specific circuitry. Here, we examine a single class of intrinsically photosensitive retinal ganglion cells and discuss their class-specific circuitry, as well as the cellular and molecular mechanisms that govern assembly of this circuitry.

Functional diversity of melanopsins and their global expression in the teleost retina.

Melanopsin (OPN4) is an opsin photopigment that, in mammals, confers photosensitivity to retinal ganglion cells and regulates circadian entrainment and pupil constriction. In non-mammalian species, two forms of opn4 exist, and are classified into mammalian-like (m) and non-mammalian-like (x) clades. However, far less is understood of the function of this photopigment family. Here we identify in zebrafish five melanopsins (opn4m-1, opn4m-2, opn4m-3, opn4x-1 and opn4x-2), each encoding a full-length opsin G protein. All five genes are expressed in the adult retina in a largely non-overlapping pattern, as revealed by RNA in situ hybridisation and immunocytochemistry, with at least one melanopsin form present in all neuronal cell types, including cone photoreceptors. This raises the possibility that the teleost retina is globally light sensitive. Electrophysiological and spectrophotometric studies demonstrate that all five zebrafish melanopsins encode a functional photopigment with peak spectral sensitivities that range from 470 to 484nm, with opn4m-1 and opn4m-3 displaying invertebrate-like bistability, where the retinal chromophore interchanges between cis- and trans-isomers in a light-dependent manner and remains within the opsin binding pocket. In contrast, opn4m-2, opn4x-1 and opn4x-2 are monostable and function more like classical vertebrate-like photopigments, where the chromophore is converted from 11-cis to all-trans retinal upon absorption of a photon, hydrolysed and exits from the binding pocket of the opsin. It is thought that all melanopsins exhibit an invertebrate-like bistability biochemistry. Our novel findings, however, reveal the presence of both invertebrate-like and vertebrate-like forms of melanopsin in the teleost retina, and indicate that photopigment bistability is not a universal property of the melanopsin family. The functional diversity of these teleost melanopsins, together with their widespread expression pattern within the retina, suggests that melanopsins confer global photosensitivity to the teleost retina and might allow for direct "fine-tuning" of retinal circuitry and physiology in the dynamic light environments found in aquatic habitats.

Celsr3 Is Required for Normal Development of GABA Circuits in the Inner Retina

The identity of the specific molecules required for the process of retinal circuitry formation is largely unknown. Here we report a newly identified zebrafish mutant in which the absence of the atypical cadherin, Celsr3, leads to a specific defect in the development of GABAergic signaling in the inner retina. This mutant lacks an optokinetic response (OKR), the ability to visually track rotating illuminated stripes, and develops a super-normal b-wave in the electroretinogram (ERG). We find that celsr3 mRNA is abundant in the amacrine and ganglion cells of the retina, however its loss does not affect synaptic lamination within the inner plexiform layer (IPL) or amacrine cell number. We localize the ERG defect pharmacologically to a late-stage disruption in GABAergic modulation of ON-bipolar cell pathway and find that the DNQX-sensitive fast b1 component of the ERG is specifically affected in this mutant. Consistently, we find an increase in GABA receptors on mutant ON-bipolar terminals, providing a direct link between the observed physiological changes and alterations in GABA signaling components. Finally, using blastula transplantation, we show that the lack of an OKR is due, at least partially, to Celsr3-mediated defects within the brain. These findings support the previously postulated inner retina origin for the b1 component and reveal a new role for Celsr3 in the normal development of ON visual pathway circuitry in the inner retina.

Retinal remodeling in the Tg P347L rabbit, a large‐eye model of retinal degeneration

Retinitis pigmentosa (RP) is an inherited blinding disease characterized by progressive loss of retinal photoreceptors. There are numerous rodent models of retinal degeneration, but most are poor platforms for interventions that will translate into clinical practice. The rabbit possesses a number of desirable qualities for a model of retinal disease including a large eye and an existing and substantial knowledge base in retinal circuitry, anatomy, and ophthalmology. We have analyzed degeneration, remodeling, and reprogramming in a rabbit model of retinal degeneration, expressing a rhodopsin proline 347 to leucine transgene in a TgP347L rabbit as a powerful model to study the pathophysiology and treatment of retinal degeneration. We show that disease progression in the TgP347L rabbit closely tracks human cone-sparing RP, including the cone-associated preservation of bipolar cell signaling and triggering of reprogramming. The relatively fast disease progression makes the TgP347L rabbit an excellent model for gene therapy, cell biological intervention, progenitor cell transplantation, surgical interventions, and bionic prosthetic studies.

Switching on the Lights: The Use of Optogenetics to Advance Retinal Gene Therapy

Photoreceptors carry a high number of mitochondria necessary to maintain their high metabolic rate. This allows them to respond rapidly to small changes in illumination. However, the metabolically demanding role of light reception appears to render photoreceptors particularly vulnerable to mutations; this is manifested in the many clinical forms of hereditary retinal degeneration (RD).1 Remarkably, in many cases the remainder of the retinal circuitry remains capable of firing despite the death of the photoreceptor cells.2,3 In this issue of Molecular Therapy, Caporale et al. exploit this residual capacity for signal transmission so as to endow retinal ganglion cells with the ability to take over the task of light detection from the photoreceptors in conditions of RD.4

Direction-Selective Circuitry in Rat Retina Develops Independently of GABAergic, Cholinergic and Action Potential Activity

The ON-OFF direction selective ganglion cells (DSGCs) in the mammalian retina code image motion by responding much more strongly to movement in one direction. They do so by receiving inhibitory inputs selectively from a particular sector of processes of the overlapping starburst amacrine cells, a type of retinal interneuron. The mechanisms of establishment and regulation of this selective connection are unknown. Here, we report that in the rat retina, the morphology, physiology of the ON-OFF DSGCs and the circuitry for coding motion directions develop normally with pharmacological blockade of GABAergic, cholinergic activity and/or action potentials for over two weeks from birth. With recent results demonstrating light independent formation of the retinal DS circuitry, our results strongly suggest the formation of the circuitry, i.e., the connections between the second and third order neurons in the visual system, can be genetically programmed, although emergence of direction selectivity in the visual cortex appears to require visual experience.

Developmental time course distinguishes changes in spontaneous and light-evoked retinal ganglion cell activity in rd1 and rd10 mice

In a subset of hereditary retinal diseases, early photoreceptor degeneration causes rapidly progressive blindness in children. To better understand how retinal development may interact with degenerative processes, we compared spontaneous and light-evoked activity among retinal ganglion cells in rd1 and rd10 mice, strains with closely related retinal disease. In each, a mutation in the Pde6b gene causes photoreceptor dysfunction and death, but in rd10 mice degeneration starts after a peak in developmental plasticity of retinal circuitry and thereafter progresses more slowly. In vitro multielectrode action potential recordings revealed that spontaneous waves of correlated ganglion cell activity comparable to those in wild-type mice were present in rd1 and rd10 retinas before eye opening [postnatal day (P) 7 to P8]. In both strains, spontaneous firing rates increased by P14-P15 and were many times higher by 4-6 wk of age. Among rd1 ganglion cells, all responses to light had disappeared by ~P28, yet in rd10 retinas vigorous ON and OFF responses were maintained well beyond this age and were not completely lost until after P60. This difference in developmental time course separates mechanisms underlying the hyperactivity from those that alter light-driven responses in rd10 retinas. Moreover, several broad physiological groups of cells remained identifiable according to response polarity and time course as late as P60. This raises hope that visual function might be preserved or restored despite ganglion cell hyperactivity seen in inherited retinal degenerations, particularly if treatment or manipulation of early developmental plasticity were to be timed appropriately.

Inner and outer retinal mechanisms engaged by epiretinal stimulation in normal and rd mice

Retinal prosthetic devices are being developed to bypass degenerated retinal photoreceptors by directly activating retinal neurons with electrical stimulation. However, the retinal circuitry that is activated by epiretinal stimulation is not well characterized. Whole-cell patch clamp recordings were obtained from ganglion cells in normal and rd mice using flat-mount and retinal slice preparations. A stimulating electrode was positioned along the ganglion cell side of the preparation at different distances from the stimulated tissue. Pulses of cathodic current evoked action potentials in ganglion cells and less frequently evoked sustained inward currents that appeared synaptic in origin. Sustained currents reversed around E(Cl) and were inhibited by blockade of α-amino-3-hydroxyl-5-methyl-4-isoxazole-proprionate (AMPA)-type glutamate receptors with 2,3-dihydroxy-6-nitro-sulfamoyl-benzo(f)-quinoxaline-2,3-dione (NBQX), γ aminobutyric acid a/c (GABA(a/c)) receptors with picrotoxinin, or glycine receptors with strychnine. This suggests that epiretinal stimulation activates glutamate release from bipolar cell terminals, which in turn evokes release of GABA and glycine from amacrine cells. Synaptic current thresholds were lower in ON ganglion cells than OFF cells, but the modest difference did not attain statistical significance. Synaptic currents were rarely observed in rd mice lacking photoreceptors compared to normal retina. In addition, confocal calcium imaging experiments in normal mice retina slices revealed that epiretinal stimulation evoked calcium increases in the outer plexiform layer. These results imply a contribution from photoreceptor inputs to the synaptic currents observed in ganglion cells. The paucity of synaptic responses in rd mice retina slices suggests that it is better to target retinal ganglion cells directly rather than to attempt to engage the inner retinal circuitry.

Imaging light responses of targeted neuron populations in the rodent retina.

Decoding the wiring diagram of the retina requires simultaneous observation of activity in identified neuron populations. Available recording methods are limited in their scope: electrodes can access only a small fraction of neurons at once, whereas synthetic fluorescent indicator dyes label tissue indiscriminately. Here, we describe a method for studying retinal circuitry at cellular and subcellular levels combining two-photon microscopy and a genetically encoded calcium indicator. Using specific viral and promoter constructs to drive expression of GCaMP3, we labeled all five major neuron classes in the adult mouse retina. Stimulus-evoked GCaMP3 responses as imaged by two-photon microscopy permitted functional cell type annotation. Fluorescence responses were similar to those measured with the small molecule dye OGB-1. Fluorescence intensity correlated linearly with spike rates >10 spikes/s, and a significant change in fluorescence always reflected a significant change in spike firing rate. GCaMP3 expression had no apparent effect on neuronal function. Imaging at subcellular resolution showed compartment-specific calcium dynamics in multiple identified cell types.

Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina

In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells, and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL)1–3: a laminar region that is conventionally divided into five major parallel sublaminae1,2. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here, we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs), and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo with respect to the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.

Differential Targeting of Optical Neuromodulators to Ganglion Cell Soma and Dendrites Allows Dynamic Control of Center-Surround Antagonism

Retinal degenerative diseases cause photoreceptor loss and often result in remodeling and deafferentation of the inner retina. Fortunately, ganglion cell morphology appears to remain intact long after photoreceptors and distal retinal circuitry have degenerated. We have introduced the optical neuromodulators channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR) differentially into the soma and dendrites of ganglion cells to recreate antagonistic center-surround receptive field interactions. We then reestablished the physiological receptive field dimensions of primate parafoveal ganglion cells by convolving Gaussian-blurred versions of the visual scene at the appropriate wavelength for each neuromodulator with the Gaussians inherent in the soma and dendrites. These Gaussian-modified ganglion cells responded with physiologically relevant antagonistic receptive field components and encoded edges with parafoveal resolution. This approach bypasses the degenerated areas of the distal retina and could provide a first step in restoring sight to individuals suffering from retinal disease.

Different Inner Retinal Pathways Mediate Rod-Cone Input in Irradiance Detection for the Pupillary Light Reflex and Regulation of Behavioral State in Mice

Detection of light in the eye contributes both to spatial awareness (form vision) and to responses that acclimate an animal to gross changes in light (irradiance detection). This dual role means that eye disease that disrupts form vision can also adversely affect physiology and behavioral state. The purpose of this study was to investigate how inner retinal circuitry mediating rod-cone photoreceptor input contributes to functionally distinct irradiance responses and whether that might account for phenotypic diversity in retinal disease. The sensitivity of the pupillary light reflex and negative masking (activity suppression by light) was measured in wild-type mice with intact inner retinal circuitry, Nob4 mice that lack ON-bipolar cell function, and rd1 mice that lack rods and cones and, therefore, have no input to ON or OFF bipolar cells. An expected increase in sensitivity to negative masking with loss of photoreceptor input in rd1 was duplicated in Nob4 mice. In contrast, sensitivity of the pupillary light reflex was more severely reduced in rd1 than in Nob4 mice. Absence of ON-bipolar cell-mediated rod-cone input can fully explain the phenotype of outer retina degeneration for negative masking but not for the pupillary light reflex. Therefore, inner retinal pathways mediating rod-cone input are different for negative masking and the pupillary light reflex.

Bipolar cells of the ground squirrel retina

Parallel processing of an image projected onto the retina starts at the first synapse, the cone pedicle, and each cone feeds its light signal into a minimum of eight different bipolar cell types. Hence, the morphological classification of bipolar cells is a prerequisite for analyzing retinal circuitry. Here we applied common bipolar cell markers to the cone-dominated ground squirrel retina, studied the labeling by confocal microscopy and electron microscopy, and compared the resulting bipolar cell types with those of the mouse (rod dominated) and primate retina. Eight different cone bipolar cell types (three OFF and five ON) and one rod bipolar cell were distinguished. The major criteria for classifying the cells were their immunocytochemical identity, their dendritic branching pattern, and the shape and stratification level of their axons in the inner plexiform layer (IPL). Immunostaining with antibodies against Gγ13, a marker for ON bipolar cells, made it possible to separate OFF and ON bipolars. Recoverin-positive OFF bipolar cells partly overlapped with ON bipolar axon terminals at the ON/OFF border of the IPL. Antibodies against HCN4 labeled the S-cone selective (bb) bipolar cell. The calcium-binding protein CaB5 was expressed in two OFF and two ON cone bipolar cell types, and CD15 labeled a widefield ON cone bipolar cell comparable to the DB6 in primate.

Activity-dependent disruption of intersublaminar spaces and ABAKAN expression does not impact functional on and off organization in the ferret retinogeniculate system

In the adult visual system, functionally distinct retinal ganglion cells (RGCs) within each eye project to discrete targets in the brain. In the ferret, RGCs encoding light increments or decrements project to independent On and Off sublaminae within each eye-specific layer of the dorsal lateral geniculate nucleus (dLGN). Here we report a manipulation of retinal circuitry that alters RGC action potential firing patterns during development and eliminates the anatomical markers of segregated On and Off sublaminae in the LGN, including the intersublaminar spaces and the expression of a glial-associated inhibitory molecule, ABAKAN, normally separating On and Off leaflets. Despite the absence of anatomically defined On and Off sublaminae, electrophysiological recordings in the dLGN reveal that On and Off dLGN cells are segregated normally. These data demonstrate a dissociation between normal anatomical sublamination and segregation of function in the dLGN and call into question a purported role for ABAKAN boundaries in the developing visual system.

Impairment of retinal adaptive circuitry in the myopic eye

Previous studies have proposed that the inner retina is affected in myopes. This study aimed to investigate the changes in adaptive circuitry of the inner retina in myopia, using the global flash multifocal electroretinogram (global flash mfERG) with different levels of contrast (luminance modulation). Fifty-four myopes had global flash mfERG recorded with different contrasts. The direct component (DC) and the induced component (IC) of the mfERG response were pooled into six regions for analysis. The response amplitudes and implicit times at different contrasts were also analysed. Results showed that myopes had significant reduction in the paracentral DC amplitude for the 29% and 49% contrasts and in the paracentral IC amplitude at all contrasts measured. The peripheral IC amplitude for the 49% contrast was also reduced. No significant change was found in implicit time for either DC or IC response. Refractive error explained about 14% of the variance in DC and 16% of the variance in IC amplitude respectively; axial length could not account for additional variance in either paracentral DC or IC amplitudes in the hierarchical regression models used. We concluded that the paracentral retinal region in myopes showed signs of impaired retinal adaptation, suggesting a functional loss at the inner retinal layer. In addition, functions attributed to the outer retinal layer showed only small changes due to myopia.

The split view of motion

In both fruitflies and vertebrates, signals from photoreceptor cells are immediately split into two opposing channels in the downstream neurons. This might facilitate the computation of visual motion. See Letter p.300 Towards the end of the nineteenth century, Ramon y Cajal, the founding father of neuroscience, observed similarities between the vertebrate retina and the insect eye. His conclusions were based purely on anatomy. Now, using state-of-the-art genetics and electrophysiology, Alexander Borst and colleagues confirm the similarity between vertebrate and insect retinal circuitry, this time at the functional level. They have distinguished motion-sensitive neurons responding to abrupt increases in light (ON-neurons), from those specific for light decrements (OFF-neurons), in the fruitfly Drosophila. Clearly, the selection pressure for efficient motion detection through variations in light intensity has generated similar circuitries in these evolutionarily distant species.

Nanodomain Control of Exocytosis Is Responsible for the Signaling Capability of a Retinal Ribbon Synapse

Primary sensory circuits encode both weak and intense stimuli reliably, requiring that their synapses signal over a wide dynamic range. In the retinal circuitry subserving night vision, processes intrinsic to the rod bipolar (RB) cell presynaptic active zone (AZ) permit the RB synapse to encode signals generated by the absorption of single photons as well as by more intense stimuli. In a study using an in vitro slice preparation of the mouse retina, we provide evidence that the location of Ca channels with low open probability within nanometers of the release sites is a critical determinant of the physiological behavior of the RB synapse. This gives rise to apparent one-to-one coupling between Ca channel opening and vesicle release, allowing presynaptic potential to be encoded linearly over a wide dynamic range. Further, it permits a transition from univesicular to multivesicular release (MVR) when two Ca channels/AZ open at potentials above the threshold for exocytosis. MVR permits small presynaptic voltage changes to elicit postsynaptic responses larger than quantal synaptic noise.

CELLULAR INHIBITORY BEHAVIOR UNDERLYING THE FORMATION OF RETINAL DIRECTION SELECTIVITY IN THE STARBURST NETWORK

Optical imaging of dendritic calcium signals provided evidence of starburst amacrine cells exhibiting calcium bias to somatofugal motion. In contrast, it has been impractical to use a dual-patch clamp technique to record membrane potentials from both proximal dendrites and distal varicosities of starburst amacrine cells in order to unequivocally prove that they are directionally sensitive to voltage, as was first suggested almost two decades ago. This paper aims to extend the passive cable model to an active cable model of a starburst amacrine cell that is intrinsically dependent on the electrical properties of starburst amacrine cells, whose various macroscopic currents are described quantitatively. The coupling between voltage and calcium just below the membrane results in a voltage-calcium system of coupled nonlinear Volterra integral equations whose solutions must be integrated into a prescribed model for example, for a synaptic couplet of starburst amacrine cells. Networks of starburst amacrine cells play a fundamental role in the retinal circuitry underlying directional selectivity. It is suggested that the dendritic plexus of starburst amacrine cells provides the substrate for the property of directional selectivity, while directional selectivity is a property of the exclusive layerings and confinement of their interconnections within the sublaminae of the inner plexiform layer involving cone bipolar cells and directionally selective ganglion cells.

Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina

Cell type-specific green fluorescent protein (GFP) expression in the retina has been achieved in an expanding repertoire of transgenic mouse lines, which are valuable tools for dissecting the retinal circuitry. However, measuring light responses from GFP-labeled cells is challenging because single-photon excitation of GFP easily bleaches photoreceptors. To circumvent this problem, we use two-photon excitation at 920 nm to target GFP-expressing cells, followed by electrophysiological recording of light responses using conventional infrared optics. This protocol offers fast and sensitive detection of GFP while preserving the light sensitivity of the retina, and can be used to obtain light responses and the detailed morphology of a GFP-expressing cell. Targeting of a GFP-expressing neuron takes less than 3 min, and the retina preparation remains light sensitive and suitable for recording for at least 8 h. This protocol can also be applied to study retinal neurons labeled with other two photon-excitable fluorophores. It is assumed that potential users of this protocol will have a basic understanding of retinal physiology and patch-clamp recording, which are not described in detail here.