Diversity of Ganglion Cell Responses to Saccade-Like Image Shifts in the Primate Retina.

Saccades are a fundamental part of natural vision. They interrupt fixations of the visual gaze and rapidly shift the image that falls onto the retina. These stimulus dynamics can cause activation or suppression of different retinal ganglion cells, but how they affect the encoding of visual information in different types of ganglion cells is largely unknown. Here, we recorded spiking responses to saccade-like shifts of luminance gratings from ganglion cells in isolated marmoset retinas and investigated how the activity depended on the combination of presaccadic and postsaccadic images. All identified cell types, On and Off parasol and midget cells, as well as a type of Large Off cells, displayed distinct response patterns, including particular sensitivity to either the presaccadic or the postsaccadic image or combinations thereof. In addition, Off parasol and Large Off cells, but not On cells, showed pronounced sensitivity to whether the image changed across the transition. Stimulus sensitivity of On cells could be explained based on their responses to step changes in light intensity, whereas Off cells, in particular, parasol and the Large Off cells, seem to be affected by additional interactions that are not triggered during simple light-intensity flashes. Together, our data show that ganglion cells in the primate retina are sensitive to different combinations of presaccadic and postsaccadic visual stimuli. This contributes to the functional diversity of the output signals of the retina and to asymmetries between On and Off pathways and provides evidence of signal processing beyond what is triggered by isolated steps in light intensity.SIGNIFICANCE STATEMENT Sudden eye movements (saccades) shift our direction of gaze, bringing new images in focus on our retinas. To study how retinal neurons deal with these rapid image transitions, we recorded spiking activity from ganglion cells, the output neurons of the retina, in isolated retinas of marmoset monkeys while shifting a projected image in a saccade-like fashion across the retina. We found that the cells do not just respond to the newly fixated image, but that different types of ganglion cells display different sensitivities to the presaccadic and postsaccadic stimulus patterns. Certain Off cells, for example, are sensitive to changes in the image across transitions, which contributes to differences between On and Off information channels and extends the range of encoded stimulus features.

Optogenetic restoration of high sensitivity vision with bReaChES, a red-shifted channelrhodopsin.

The common final pathway to blindness in many forms of retinal degeneration is the death of the light-sensitive primary retinal neurons. However, the normally light-insensitive second- and third-order neurons persist optogenetic gene therapy aims to restore sight by rendering such neurons light-sensitive. Here, we investigate whether bReaChES, a newly described high sensitivity Type I opsin with peak sensitivity to long-wavelength visible light, can restore vision in a murine model of severe early-onset retinal degeneration. Intravitreal injection of an adeno-associated viral vector carrying the sequence for bReaChES downstream of the calcium calmodulin kinase IIα promoter resulted in sustained retinal expression of bReaChES. Retinal ganglion cells (RGCs) expressing bReaChES generated action potentials at light levels consistent with bright indoor lighting (from 13.6 log photons cm-2 s-1). They could also detect flicker at up to 50 Hz, which approaches the upper temporal limit of human photopic vision. Topological response maps of bReaChES-expressing RGCs suggest that optogenetically activated RGCs may demonstrate similar topographical responses to RGCs stimulated by photoreceptor activation. Furthermore, treated dystrophic mice displayed restored cortical neuronal activity in response to light and rescued behavioral responses to a looming stimulus that simulated an aerial predator. Finally, human surgical retinal explants exposed to the bReaChES treatment vector demonstrated transduction. Together, these findings suggest that intravitreal gene therapy to deliver bReaChES to the retina may restore vision in human retinal degeneration in vivo at ecologically relevant light levels with spectral and temporal response characteristics approaching those of normal human photopic vision.

Tonic Endocannabinoid Levels Modulate Retinal Signaling.

The endocannabinoid (eCB) system is critically involved in the modulation of synaptic transmission in the central nervous system, playing an important role in the control of emotional responses, neurodevelopment and synaptic plasticity among other functions. The eCB system is also present in the retina, with studies indicating changes in function after application of cannabinoid receptor agonists, antagonists and in knockout models. Whether eCBs are tonically released in the retina and their physiological functions is, however, still unknown. We investigated the role of the eCB system in the modulation of response strength of retinal ganglion cells (RGCs) to light stimulation, their receptive field organization, contrast sensitivity and excitability properties by performing whole-cell patch-clamp recordings in mouse RGCs before and after bath application of URB597, an inhibitor of the enzyme that degrades the eCB anandamide. Our results show that URB597 application leads to a reduction in the strength of synaptic inputs onto RGCs but paradoxically increases RGC excitability. In addition, URB597 was shown to modulate receptive field organization and contrast sensitivity of RGCs. We conclude that tonically released eCBs modulate retinal signaling by acting on traditional cannabinoid receptors (CB1R/CB2R) as well as on non-cannabinoid receptor targets. Thus, a thorough understanding of the effects of drugs that alter the endogenous cannabinoid levels and of exogenous cannabinoids is necessary to fully comprehend the impact of their medical as well as recreational use on vision.

Rapid Analysis of Visual Receptive Fields by Iterative Tomography.

Many receptive fields in the early visual system show standard (center-surround) structure and can be analyzed using simple drifting patterns and a difference-of-Gaussians (DoG) model, which treats the receptive field as a linear filter of the visual image. But many other receptive fields show nonlinear properties such as selectivity for direction of movement. Such receptive fields are typically studied using discrete stimuli (moving or flashed bars and edges) and are modelled according to the features of the visual image to which they are most sensitive. Here, we harness recent advances in tomographic image analysis to characterize rapidly and simultaneously both the linear and nonlinear components of visual receptive fields. Spiking and intracellular voltage potential responses to briefly flashed bars are analyzed using non-negative matrix factorization (NNMF) and iterative reconstruction tomography (IRT). The method yields high-resolution receptive field maps of individual neurons and neuron ensembles in primate (marmoset, both sexes) lateral geniculate and rodent (mouse, male) retina. We show that the first two IRT components correspond to DoG-equivalent center and surround of standard [magnocellular (M) and parvocellular (P)] receptive fields in primate geniculate. The first two IRT components also reveal the spatiotemporal receptive field structure of nonstandard (on/off-rectifying) receptive fields. In rodent retina we combine NNMF-IRT with patch-clamp recording and dye injection to directly map spatial receptive fields to the underlying anatomy of retinal output neurons. We conclude that NNMF-IRT provides a rapid and flexible framework for study of receptive fields in the early visual system.

The temporal structure of the inner retina at a single glance.

The retina decomposes visual stimuli into parallel channels that encode different features of the visual environment. Central to this computation is the synaptic processing in a dense layer of neuropil, the so-called inner plexiform layer (IPL). Here, different types of bipolar cells stratifying at distinct depths relay the excitatory feedforward drive from photoreceptors to amacrine and ganglion cells. Current experimental techniques for studying processing in the IPL do not allow imaging the entire IPL simultaneously in the intact tissue. Here, we extend a two-photon microscope with an electrically tunable lens allowing us to obtain optical vertical slices of the IPL, which provide a complete picture of the response diversity of bipolar cells at a "single glance". The nature of these axial recordings additionally allowed us to isolate and investigate batch effects, i.e. inter-experimental variations resulting in systematic differences in response speed. As a proof of principle, we developed a simple model that disentangles biological from experimental causes of variability and allowed us to recover the characteristic gradient of response speeds across the IPL with higher precision than before. Our new framework will make it possible to study the computations performed in the central synaptic layer of the retina more efficiently.

Cannabinoid-induced increase of quantal size and enhanced neuromuscular transmission.

Cannabinoids exert dynamic control over many physiological processes including memory formation, cognition and pain perception. In the central nervous system endocannabinoids mediate negative feedback of quantal transmitter release following postsynaptic depolarization. The influence of cannabinoids in the peripheral nervous system is less clear and might have broad implications for the therapeutic application of cannabinoids. We report a novel cannabinoid effect upon the mouse neuromuscular synapse: acutely increasing synaptic vesicle volume and raising the quantal amplitudes. In a mouse model of myasthenia gravis the cannabinoid receptor agonist WIN 55,212 reversed fatiguing failure of neuromuscular transmission, suggesting future therapeutic potential. Our data suggest an endogenous pathway by which cannabinoids might help to regulate transmitter release at the neuromuscular junction.

The impact of inhibitory mechanisms in the inner retina on spatial tuning of RGCs.

Spatial tuning properties of retinal ganglion cells (RGCs) are sharpened by lateral inhibition originating at both the outer and inner plexiform layers. Lateral inhibition in the retina contributes to local contrast enhancement and sharpens edges. In this study, we used dynamic clamp recordings to examine the contribution of inner plexiform inhibition, originating from spiking amacrine cells, to the spatial tuning of RGCs. This was achieved by injecting currents generated from physiologically recorded excitatory and inhibitory stimulus-evoked conductances, into different types of primate and mouse RGCs. We determined the effects of injections of size-dependent conductances in which presynaptic inhibition and/or direct inhibition onto RGCs were partly removed by blocking the activity of spiking amacrine cells. We found that inhibition originating from spiking amacrine cells onto bipolar cell terminals and onto RGCs, work together to sharpen the spatial tuning of RGCs. Furthermore, direct inhibition is crucial for preventing spike generation at stimulus offset. These results reveal how inhibitory mechanisms in the inner plexiform layer contribute to determining size tuning and provide specificity to stimulus polarity.

Vestibular Interactions in the Thalamus.

It has long been known that the vast majority of all information en route to the cerebral cortex must first pass through the thalamus. The long held view that the thalamus serves as a simple hi fidelity relay station for sensory information to the cortex, however, has over recent years been dispelled. Indeed, multiple projections from the vestibular nuclei to thalamic nuclei (including the ventrobasal nuclei, and the geniculate bodies)- regions typically associated with other modalities- have been described. Further, some thalamic neurons have been shown to respond to stimuli presented from across sensory modalities. For example, neurons in the rat anterodorsal and laterodorsal nuclei of the thalamus respond to visual, vestibular, proprioceptive and somatosensory stimuli and integrate this information to compute heading within the environment. Together, these findings imply that the thalamus serves crucial integrative functions, at least in regard to vestibular processing, beyond that imparted by a "simple" relay. In this mini review we outline the vestibular inputs to the thalamus and provide some clinical context for vestibular interactions in the thalamus. We then focus on how vestibular inputs interact with other sensory systems and discuss the multisensory integration properties of the thalamus.

Characterization of secretagogin-immunoreactive amacrine cells in marmoset retina.

The retina contains at least 30 different types of amacrine cells but not many are well characterized. In the present study the calcium-binding protein secretagogin was localized in a population of regular and displaced amacrine cells in the retina of the common marmoset Callithrix jacchus. Irrespective of their soma location, the dendrites of secretagogin amacrine cells occupy strata 2, 3, and 4 of the inner plexiform layer, between the two bands formed by cholinergic amacrine cells. Segretagogin amacrine cells are also immunopositive to antibodies against glutamic acid decarboxylase, suggesting that they use γ-aminobutyric acid (GABA) as their neurotransmitter. The spatial density of secretagogin amacrine cells decreases from a peak of about 400 cells/mm(2) near 1 mm eccentricity to less than 100 cells/mm(2) in peripheral retina; these densities account for about 1% of amacrine cells in the inner nuclear layer and for up to 27% of displaced amacrine cells. The cell bodies form a regular mosaic, suggesting that they constitute a single amacrine cell population. Secretagogin cells have varicose dendrites, which are decorated with small spines. Intracellular injection of DiI into secretagogin cells revealed an average dendritic field diameter of 170 µm and an average coverage factor of 3.2. In summary, secretagogin cells in marmoset retina are medium-field amacrine cells that share their stratification pattern with narrow-field amacrine cells and their neurotransmitter with wide-field amacrine cells. They may mediate spatial inhibition spanning the centralmost (on and off) bands of the inner plexiform layer.

Excitatory and inhibitory contributions to receptive fields of alpha-like retinal ganglion cells in mouse.

The ON and OFF pathways that emerge at the first synapse in the retina are generally thought to be streamed in parallel to higher visual areas, but recent work shows cross talk at the level of retinal ganglion cells. The ON pathway drives inhibitory inputs onto some OFF ganglion cells, such that these neurons show "push-pull" convergence of OFF-excitation and ON-disinhibition. In this study we measure the spatial receptive field of excitatory and inhibitory inputs to OFF-sustained (OFF-S) retinal ganglion cells of mouse, establish how contrast adaptation modulates excitatory and inhibitory synaptic inputs, and show the pharmacology of the inhibitory inputs. We find that the spatial tuning properties of excitatory and inhibitory inputs are sufficient to determine the spatial profile of the spike output and that high spatial acuity may be particularly reliant on disinhibitory circuits. Contrast adaptation reduced excitation to OFF-S ganglion cells, as expected, and also unmasked an asymmetry in inhibitory inputs: disinhibition at light-off was immune to contrast adaptation, but inhibition at light-on was substantially reduced. In pharmacological experiments we confirm that inhibitory inputs are partly mediated by glycine, but our measurements also suggest a substantial role for GABA. Our observations therefore reveal functional diversity in the inhibitory inputs to OFF ganglion cells and suggest that in addition to enhancing operational range these inputs help shape the spatial receptive fields of ganglion cells.

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.

Responses of retinal ganglion cells to extracellular electrical stimulation, from single cell to population: model-based analysis.

Retinal ganglion cells (RGCs), which survive in large numbers following neurodegenerative diseases, could be stimulated with extracellular electric pulses to elicit artificial percepts. How do the RGCs respond to electrical stimulation at the sub-cellular level under different stimulus configurations, and how does this influence the whole-cell response? At the population level, why have experiments yielded conflicting evidence regarding the extent of passing axon activation? We addressed these questions through simulations of morphologically and biophysically detailed computational RGC models on high performance computing clusters. We conducted the analyses on both large-field RGCs and small-field midget RGCs. The latter neurons are unique to primates. We found that at the single cell level the electric potential gradient in conjunction with neuronal element excitability, rather than the electrode center location per se, determined the response threshold and latency. In addition, stimulus positioning strongly influenced the location of RGC response initiation and subsequent activity propagation through the cellular structure. These findings were robust with respect to inhomogeneous tissue resistivity perpendicular to the electrode plane. At the population level, RGC cellular structures gave rise to low threshold hotspots, which limited axonal and multi-cell activation with threshold stimuli. Finally, due to variations in neuronal element excitability over space, following supra-threshold stimulation some locations favored localized activation of multiple cells, while others favored axonal activation of cells over extended space.

Permanent functional reorganization of retinal circuits induced by early long-term visual deprivation.

Early sensory experience shapes the functional and anatomical connectivity of neuronal networks. Light deprivation alters synaptic transmission and modifies light response properties in the visual system, from retinal circuits to higher visual centers. These effects are more pronounced during a critical period in juvenile life and are mostly reversed by restoring normal light conditions. Here we show that complete light deprivation, from birth to periods beyond the critical period, permanently modifies the receptive field properties of retinal ganglion cells. Visual deprivation reduced both the strength of light responses in ganglion cells and their receptive field size. Light deprivation produced an imbalance in the ratio of inhibitory to excitatory inputs, with a shift toward larger inhibitory conductances. Ganglion cell receptive fields in visually deprived animals showed a spatial mismatch of inhibitory and excitatory inputs and inhibitory inputs were highly scattered over the receptive field. These results indicate that visual experience early in life is critical for the refinement of retinal circuits and for appropriate signaling of the spatiotemporal properties of visual stimuli, thus influencing the response properties of neurons in higher visual centers and their processing of visual information.

Ionotropic glutamate receptors of amacrine cells of the mouse retina.

The mammalian retina contains approximately 30 different morphological types of amacrine cells, receiving glutamatergic input from bipolar cells. In this study, we combined electrophysiological and pharmacological techniques in order to study the glutamate receptors expressed by different types of amacrine cells. Whole-cell currents were recorded from amacrine cells in vertical slices of the mouse retina. During the recordings the cells were filled with Lucifer Yellow/Neurobiotin allowing classification as wide-field or narrow-field amacrine cells. Amacrine cell recordings were also carried out in a transgenic mouse line whose glycinergic amacrine cells express enhanced green fluorescent protein (EGFP). Agonist-induced currents were elicited by exogenous application of NMDA, AMPA, and kainate (KA) while holding cells at -75 mV. Using a variety of specific agonists and antagonists (NBQX, AP5, cyclothiazide, GYKI 52466, GYKI 53655, SYM 2081) responses mediated by AMPA, KA, and NMDA receptors could be dissected. All cells (n = 300) showed prominent responses to non-NMDA agonists. Some cells expressed AMPA receptors exclusively and some cells expressed KA receptors exclusively. In the majority of cells both receptor types could be identified. NMDA receptors were observed in about 75% of the wide-field amacrine cells and in less than half of the narrow-field amacrine cells. Our results confirm that different amacrine cell types express distinct sets of ionotropic glutamate receptors, which may be critical in conferring their unique temporal responses to this diverse neuronal class.

Light signaling in scotopic conditions in the rabbit, mouse and rat retina: a physiological and anatomical study.

In the dark, light signals are conventionally routed through the following circuit: rods synapse onto rod bipolar (RB) cells, which in turn contact AII amacrine cells. AII cells segregate the light signal into the on and off pathways by making electrical synapses with on cone bipolar (CB) cells and glycinergic inhibitory chemical synapses with off CB cells. These bipolar cells synapse onto their respective ganglion cells, which transfer on and off signals to the visual centers of the brain. Two alternative pathways have recently been postulated for the signal transfer in scotopic conditions: 1) electrical coupling between rods and cones, and 2) a circuit independent of cone photoreceptors, implying direct contacts between rods and off CB cells. Anatomical evidence supports the existence of both these circuits. To investigate the contribution of these alternative pathways to scotopic vision in the mammalian retina, we have performed patch-clamp recordings from ganglion cells in the dark-adapted retina of the rabbit, mouse, and rat. Approximately one-half of the ganglion cells in the rabbit retina received off signals through a circuit that was independent of RB cells. This was shown by their persistence in the presence of the glutamate agonist 2-amino-4-phosphonobutyric acid (APB), which blocks rod-->RB cell signaling. Consistent with this result, strychnine, a glycine receptor antagonist, was unable to abolish these off responses. In addition, we were able to show that some off cone bipolar dendrites terminate at rod spherules and make potential contacts. In the mouse retina, however, there seems to be a very low proportion of off signals carried by an APB-resistant pathway. No ganglion cells in the rat retina displayed APB- and strychnine-resistant responses. Our data support signaling through flat contacts between rods and off CB cells as the alternative route, but suggest that the significance of this pathway differs between species.

AII amacrine cells express L-type calcium channels at their output synapses.

AII amacrine cells play a critical role in the high-fidelity signal transmission pathways involved with nighttime vision. The temporal properties of the light responses strongly depend on the transfer function at different synaptic stages and consequently on presynaptic calcium influx. AII light responses are complex waveforms generated by graded input, they comprise Na+-based spikes as well as a sustained component, and they are transferred to graded cone bipolar cells. It is, therefore, of interest to determine the properties of AII voltage-dependent calcium channels (VDCCs) to establish whether these cells express N-type and/or P/Q-type VDCCs, characteristic of spiking neurons, or whether they are more like graded neurons, which mostly use L-type VDCCs. We combined electrophysiological, molecular biological, and imaging techniques to characterize calcium currents and their sites of origin in mouse AII amacrine cells. Calcium currents activated at potentials more positive than -60 mV (maximally between -50 and -20 mV) and inactivated slowly. These currents were blocked by dihydropyridine (DHP) antagonists and were enhanced by the DHP agonist BayK 8644. Single-cell RT-PCR analysis of mRNA encoding for different calcium channel alpha subunits in AIIs revealed a consistent expression of the alpha1-D subunit. Calcium imaging of AII cells showed that the greatest change in intracellular calcium occurred in the lobular appendages, with minor changes being observed in the arboreal dendrites. Depolarization-induced calcium rises were also modulated by DHPs, suggesting that a particular kind of L-type VDCC, mainly localized to the lobular appendages, enables these spiking-capable neurons to release neurotransmitter in a sustained manner onto OFF-cone bipolar cells.