A common cause for nystagmus in different congenital stationary night blindness mouse models.

In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), synchronous oscillating retinal ganglion cells (RGCs) lead to oscillatory eye movements, i.e. nystagmus. Given the specific expression of mGluR6 and Cav 1.4 in the photoreceptor to bipolar cell synapses, as well as their clinical association with CSNB, we hypothesize that Grm6nob3 and Cav 1.4-KO mutants show, like the Nyxnob mouse, oscillations in both their RGC activity and eye movements. Using multi-electrode array recordings of RGCs and measurements of the eye movements, we demonstrate that Grm6nob3 and Cav 1.4-KO mice also show oscillations of their RGCs as well as a nystagmus. Interestingly, the preferred frequencies of RGC activity as well as the eye movement oscillations of the Grm6nob3 , Cav 1.4-KO and Nyxnob mice differ among mutants, but the neuronal activity and eye movement behaviour within a strain remain aligned in the same frequency domain. Model simulations indicate that mutations affecting the photoreceptor-bipolar cell synapse can form a common cause of the nystagmus of CSNB by driving oscillations in RGCs via AII amacrine cells. KEY POINTS: In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), their oscillatory eye movements (i.e. nystagmus) are caused by synchronous oscillating retinal ganglion cells. Here we show that the same mechanism applies for two other CSNB mouse models - Grm6nob3 and Cav 1.4-KO mice. We propose that the retinal ganglion cell oscillations originate in the AII amacrine cells. Model simulations show that by only changing the input to ON-bipolar cells, all phenotypical differences between the various genetic mouse models can be reproduced.

Receptive Field Sizes of Nyxnob Mouse Retinal Ganglion Cells.

Patients with congenital nystagmus, involuntary eye movements, often have a reduced visual acuity. Some of these patients have a retinal-specific mutation in the protein nyctalopin, which is also present in the Nyxnob mouse. In these mice, retinal ganglion cells (RGCs) have oscillatory activity, which leads to expanded axonal projections towards the dLGN and consequently to a desegregation of retinal projections to the brain. In this study, we investigate whether the receptive fields of Nyxnob RGCs have also expanded by measuring the size of their receptive fields using MEA recordings. Contrary to our expectation, relative to wild-type (WT) mice we found receptive field sizes in the Nyxnob retina had not increased but instead had decreased for green-light preferring RGCs. Additionally, we also found the receptive fields of UV-light preferring RGCs are larger than green-light preferring RGCs in both WT and Nyxnob mice.

Enhancing the dark side: asymmetric gain of cone photoreceptors underpins their discrimination of visual scenes based on skewness.

Psychophysical data indicate that humans can discriminate visual scenes based on their skewness, i.e. the ratio of dark and bright patches within a visual scene. It has also been shown that at a phenomenological level this skew discrimination is described by the so-called blackshot mechanism, which accentuates strong negative contrasts within a scene. Here, we present a set of observations suggesting that the underlying computation might start as early as the cone phototransduction cascade, whose gain is higher for strong negative contrasts than for strong positive contrasts. We recorded from goldfish cone photoreceptors and found that the asymmetry in the phototransduction gain leads to responses with larger amplitudes when using negatively rather than positively skewed light stimuli. This asymmetry in amplitude was present in the cone photocurrent, voltage response and synaptic output. Given that the properties of the phototransduction cascade are universal across vertebrates, it is possible that the mechanism shown here gives rise to a general ability to discriminate between scenes based only on their skewness, which psychophysical studies have shown humans can do. Thus, our data suggest the importance of non-linearity of the early photoreceptor for perception. Additionally, we found that stimulus skewness leads to a subtle change in photoreceptor kinetics. For negatively skewed stimuli, the impulse response functions of the cone peak later than for positively skewed stimuli. However, stimulus skewness does not affect the overall integration time of the cone. KEY POINTS: Humans can discriminate visual scenes based on skewness, i.e. the relative prevalence of bright and dark patches within a scene. Here, we show that negatively skewed time-series stimuli induce larger responses in goldfish cone photoreceptors than comparable positively skewed stimuli. This response asymmetry originates from within the phototransduction cascade, where gain is higher for strong negative contrasts (dark patches) than for strong positive contrasts (bright patches). Unlike the implicit assumption often contained within models of downstream visual neurons, our data show that cone photoreceptors do not simply relay linearly filtered versions of visual stimuli to downstream circuitry, but that they also emphasize specific stimulus features. Given that the phototransduction cascade properties among vertebrate retinas are mostly universal, our data imply that the skew discrimination by human subjects reported in psychophysical studies might stem from the asymmetric gain function of the phototransduction cascade.

Degenerated Cones in Cultured Human Retinas Can Successfully Be Optogenetically Reactivated.

Biblical references aside, restoring vision to the blind has proven to be a major technical challenge. In recent years, considerable advances have been made towards this end, especially when retinal degeneration underlies the vision loss such as occurs with retinitis pigmentosa. Under these conditions, optogenetic therapies are a particularly promising line of inquiry where remaining retinal cells are made into "artificial photoreceptors". However, this strategy is not without its challenges and a model system using human retinal explants would aid its continued development and refinement. Here, we cultured post-mortem human retinas and show that explants remain viable for around 7 days. Within this period, the cones lose their outer segments and thus their light sensitivity but remain electrophysiologically intact, displaying all the major ionic conductances one would expect for a vertebrate cone. We optogenetically restored light responses to these quiescent cones using a lentivirus vector constructed to express enhanced halorhodopsin under the control of the human arrestin promotor. In these 'reactivated' retinas, we show a light-induced horizontal cell to cone feedback signal in cones, indicating that transduced cones were able to transmit their light response across the synapse to horizontal cells, which generated a large enough response to send a signal back to the cones. Furthermore, we show ganglion cell light responses, suggesting the cultured explant's condition is still good enough to support transmission of the transduced cone signal over the intermediate retinal layers to the final retinal output level. Together, these results show that cultured human retinas are an appropriate model system to test optogenetic vision restoration approaches and that cones which have lost their outer segment, a condition occurring during the early stages of retinitis pigmentosa, are appropriate targets for optogenetic vision restoration therapies.

High Contrast Allows the Retina to Compute More Than Just Contrast.

The goal of sensory processing is to represent the environment of an animal. All sensory systems share a similar constraint: they need to encode a wide range of stimulus magnitudes within their narrow neuronal response range. The most efficient way, exploited by even the simplest nervous systems, is to encode relative changes in stimulus magnitude rather than the absolute magnitudes. For instance, the retina encodes contrast, which are the variations of light intensity occurring in time and in space. From this perspective, it is easy to understand why the bright plumage of a moving bird gains a lot of attention, while an octopus remains motionless and mimics its surroundings for concealment. Stronger contrasts simply cause stronger visual signals. However, the gains in retinal performance associated with higher contrast are far more than what can be attributed to just a trivial linear increase in signal strength. Here we discuss how this improvement in performance is reflected throughout different parts of the neural circuitry, within its neural code and how high contrast activates many non-linear mechanisms to unlock several sophisticated retinal computations that are virtually impossible in low contrast conditions.

Nystagmus in patients with congenital stationary night blindness (CSNB) originates from synchronously firing retinal ganglion cells.

Congenital nystagmus, involuntary oscillating small eye movements, is commonly thought to originate from aberrant interactions between brainstem nuclei and foveal cortical pathways. Here, we investigated whether nystagmus associated with congenital stationary night blindness (CSNB) results from primary deficits in the retina. We found that CSNB patients as well as an animal model (nob mice), both of which lacked functional nyctalopin protein (NYX, nyx) in ON bipolar cells (BCs) at their synapse with photoreceptors, showed oscillating eye movements at a frequency of 4-7 Hz. nob ON direction-selective ganglion cells (DSGCs), which detect global motion and project to the accessory optic system (AOS), oscillated with the same frequency as their eyes. In the dark, individual ganglion cells (GCs) oscillated asynchronously, but their oscillations became synchronized by light stimulation. Likewise, both patient and nob mice oscillating eye movements were only present in the light when contrast was present. Retinal pharmacological and genetic manipulations that blocked nob GC oscillations also eliminated their oscillating eye movements, and retinal pharmacological manipulations that reduced the oscillation frequency of nob GCs also reduced the oscillation frequency of their eye movements. We conclude that, in nob mice, synchronized oscillations of retinal GCs, most likely the ON-DCGCs, cause nystagmus with properties similar to those associated with CSNB in humans. These results show that the nob mouse is the first animal model for a form of congenital nystagmus, paving the way for development of therapeutic strategies.

Silent-substitution stimuli silence the light responses of cones but not their output.

Chromatic vision starts at the retinal photoreceptors but photoreceptors are themselves color-blind, responding only to their effective quantal catch and not to the wavelength of the caught photon per se. Mitchell and Rushton (1971) termed this phenomenon the univariance concept, and it is widely used in designing silent-substitution stimuli to test the unique contributions of specific photoreceptor types to vision. In principle, this procedure controls the effective quantal catch of photoreceptors well and hence works at the phototransduction-cascade level of vision. However, both phototransduction-cascade modulation and the horizontal-cell-mediated feedback signal determine photoreceptor output. Horizontal cells receive input from, and send feedback to, more than one photoreceptor type. This should mean that silent-substitution stimuli do not silence horizontal-cell activity, and that this activity is fed back to the silenced cones. This in turn will modulate the output of silenced cones, making them not so silent after all. Here we tested this idea and found that silent-substitution stimuli can adequately silence cone-membrane potential responses. However, these cones still received a feedback signal from horizontal cells, which modulates their Ca2+ current and thus their output. These feedback-induced Ca2+-current changes are substantial, as they are of the same order of magnitude as Ca2+-current changes that occur when cones are directly stimulated with light. This illustrates that great care needs to be taken in interpreting results obtained with silent-substitution stimuli. In the discussion, we outline two basic types of interpretation pitfalls that can occur.

A novel mechanism of cone photoreceptor adaptation.

An animal's ability to survive depends on its sensory systems being able to adapt to a wide range of environmental conditions, by maximizing the information extracted and reducing the noise transmitted. The visual system does this by adapting to luminance and contrast. While luminance adaptation can begin at the retinal photoreceptors, contrast adaptation has been shown to start at later stages in the retina. Photoreceptors adapt to changes in luminance over multiple time scales ranging from tens of milliseconds to minutes, with the adaptive changes arising from processes within the phototransduction cascade. Here we show a new form of adaptation in cones that is independent of the phototransduction process. Rather, it is mediated by voltage-gated ion channels in the cone membrane and acts by changing the frequency response of cones such that their responses speed up as the membrane potential modulation depth increases and slow down as the membrane potential modulation depth decreases. This mechanism is effectively activated by high-contrast stimuli dominated by low frequencies such as natural stimuli. However, the more generally used Gaussian white noise stimuli were not effective since they did not modulate the cone membrane potential to the same extent. This new adaptive process had a time constant of less than a second. A critical component of the underlying mechanism is the hyperpolarization-activated current, Ih, as pharmacologically blocking it prevented the long- and mid- wavelength sensitive cone photoreceptors (L- and M-cones) from adapting. Consistent with this, short- wavelength sensitive cone photoreceptors (S-cones) did not show the adaptive response, and we found they also lacked a prominent Ih. The adaptive filtering mechanism identified here improves the information flow by removing higher-frequency noise during lower signal-to-noise ratio conditions, as occurs when contrast levels are low. Although this new adaptive mechanism can be driven by contrast, it is not a contrast adaptation mechanism in its strictest sense, as will be argued in the Discussion.

Extracellular ATP hydrolysis inhibits synaptic transmission by increasing ph buffering in the synaptic cleft.

Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²âº channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation may be a widespread phenomenon.

Spectacle lens compensation in the pigmented guinea pig.

When a young growing eye wears a negative or positive spectacle lens, the eye compensates for the imposed defocus by accelerating or slowing its elongation rate so that the eye becomes emmetropic with the lens in place. Such spectacle lens compensation has been shown in chicks, tree-shrews, marmosets and rhesus monkeys. We have developed a model of emmetropisation using the guinea pig in order to establish a rapid and easy mammalian model. Guinea pigs were raised with a +4D, +2D, 0D (plano), -2D or -4D lens worn in front of one eye for 10 days or a +4D on one eye and a 0D on the fellow eye for 5 days or no lens on either eye (littermate controls). Refractive error and ocular distances were measured at the end of these periods. The difference in refractive error between the eyes was linearly related to the lens-power worn. A significant compensatory response to a +4D lens occurred after only 5 days and near full compensation occurred after 10 days when the effective imposed refractive error was between 0D and 8D of hyperopia. Eyes wearing plano lenses were slightly more myopic than their fellow eyes (-1.7D) but showed no difference in ocular length. Relative to the plano group, plus and minus lenses induced relative hyperopic or myopic differences between the two eyes, inhibited or accelerated their ocular growth, and expanded or decreased the relative thickness of the choroid, respectively. In individual animals, the difference between the eyes in vitreous chamber depth and choroid thickness reached +/-100 and +/-40microm, respectively, and was significantly correlated with the induced refractive differences. Although eyes responded differentially to plus and minus lenses, the plus lenses generally corrected the hyperopia present in these young animals. The effective refractive error induced by the lenses ranged between -2D of myopic defocus to +10D of hyperopic defocus with the lens in place, and compensation was highly linear between 0D and 8D of effective hyperopic defocus, beyond which the compensation was reduced. We conclude that in the guinea pig, ocular growth and refractive error are visually regulated in a bidirectional manner to plus and minus lenses, but that the eye responds in a graded manner to imposed effective hyperopic defocus.

Emmetropization and schematic eye models in developing pigmented guinea pigs.

A model of the axial change in ocular parameters of the guinea pig eye from 2 to 825 days of age was developed and a corresponding paraxial schematic eye model applicable from 2 to 100 days of age was constructed. Axial distances increased logarithmically over time except for the lens in which growth was more complex. Over the first 30 days, ocular elongation was approximately linear: ocular length increased by 37 microm/day, the majority due lens expansion. The choroid and sclera thickened with age, while the retina thinned in proportion to the increased ocular size, and the model suggests that there is no small eye artefact for white light retinoscopy. Refractive error just after birth was +4.8D but halved within the first week. Emmetropization occurred within the first month of life similar to that in other species when aligned at the point of sexual maturity and scaled by the time taken to reach adulthood. The power of the eye was 227D at 2 days of age and reduced by 19.7D by 100 days due to a 22% decrease in the power of the cornea. The posterior nodal distance (PND) was 4.7 mm at 30 days of age, with a maximum rate of change of 13 microm/day during the first week. The ratio of PND to axial length declined until at least 100 days of age, well after emmetropia was reached. This suggests that the maintenance of emmetropia is not sustained through proportional axial growth, but involves some active mechanism beyond simple scaling. The model predicts that 1D of myopia requires an elongation of between 23 and 32 microm, depending upon age, suggesting that a resolution of at least 50 microm is required in methods used to determine the significance of ocular length changes in guinea pig models of refractive development. Retinal magnification averaged 80 microm/degree, and the maximum potential brightness of the retinal image was high, which together with a ratio of lens power to corneal power of 1.7-2.0 suggests that the guinea pig eye is adapted for nocturnal conditions.

Form-deprivation myopia in the guinea pig (Cavia porcellus).

Form deprivation (FD) was induced in 61 guinea pigs with a diffuser worn on one eye. The form-deprived eye elongated and developed myopia within 6 days in animals raised under a 12:12 h light/dark cycle, but not when reared in darkness. After 11 days of FD, the average eye was -6.6 D more myopic and 146 microm longer than its fellow eye. Initially the myopia was mostly from vitreous chamber elongation, but with longer periods of FD, corneal power increases predominated. These effects were confirmed in schematic eyes. After a delay, FD also elongated the vitreous chamber of the non-deprived eye. The myopia rapidly abated once the diffusers were removed (65% within 24 h) due to inhibition of elongation and choroidal thickening. The guinea pig provides a fast mammalian model of FD myopia and corneal curvature regulation.