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 Cav1.4 in the photoreceptor to bipolar cell synapses, as well as their clinical association with CSNB, we hypothesize that Grm6nob3 and Cav1.4-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 Cav1.4-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, Cav1.4-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. (Figure presented.). 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 Cav1.4-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.</p

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 ±40 μm, 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

HC responses in various pharmacological conditions.

<p>(A) Acidification of the extrasynaptic medium reduces the rollback response in goldfish HCs (arrow) (<i>n</i> = 4) and hyperpolarizes the HC membrane potential (see D) (black, control; red, pH 7.2; green, wash). This indicates that the slow component of feedback contributes to the rollback response in HCs. (B) Application of probenecid also led to a slight reduction of the rollback response (<i>n</i> = 18) and hyperpolarization of the HC membrane potential (see D). The rollback response never disappeared completely (black, control; red, probenecid; green, wash). (C) Application of probenecid in a Ringer's solution containing 28 mM HEPES. In this condition, the HC light responses were almost completely blocked and HC hyperpolarized strongly [see D; black, HEPES; red, HEPES+probenecid; green, HEPES (wash)]. (D) Mean ± sem HC membrane hyperpolarization induced by the various pharmacological manipulations.</p

Purinergic receptor activation does not underlie feedback from HCs to cones.

<p>(A) A whole-cell IV relation for cones in control condition (black) and with 100 µM ATP (red; <i>n</i> = 5). ATP does not activate a nonspecific cation conductance. (B) A whole-cell IV relation for cones in control condition (black) and 50 µM ARL67156 (red; <i>n</i> = 5). ARL67156 did not lead to the activation of a nonspecific cation conductance. (C) Mean feedback responses in control (black) and in 1 µM ZM 241385 (red; <i>n</i> = 4), a blocker of the A2 receptor. The response amplitude and kinetics of the feedback response were not significantly affected, indicating that feedback is not mediated by A2 receptors.</p

HCs release ATP.

<p>(A) Dissociated goldfish HC. This preparation consisted of about 90% of HCs. The remaining material was mostly debris. Scale bar, 20 µm. (B) The mean ± sem whole-cell IV relations of six dissociated HCs in conditions where potassium currents were inhibited by Cs. Application of 500 µM probenecid (red trace) reduces the conductance of the HCs. The green trace is the IV relation of the probenecid blocked current. This current has similar characteristics as the Panx1 current described by Prochnow et al. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001864#pbio.1001864-Prochnow1" target="_blank">[11]</a>. (C) Change in ATP release as measured with the luminescence assay. Depolarization of dissociated HCs by AMPA led to an increase in ATP release, whereas inhibition with probenecid decreased the release significantly. In probenecid, the ATP release was significantly lower than baseline, indicating that HCs in control conditions release ATP.</p

Pharmacological profile of the slow component of feedback.

<p>(A) Normalized mean feedback responses of nine cones in control (black) and in 500 µM probenecid (red). The slow component present in control conditions was reduced by probenecid. (B) Normalized mean feedback responses in nine cones in control (black) and in 100 µM ATP (red). ATP blocked the slow component of feedback, indicating the slow component depends on ATP. Note this concentration of ATP does not block Panx1 channels <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001864#pbio.1001864-Kurtenbach1" target="_blank">[20]</a>. (C) Normalized mean feedback responses in seven cones in control (black) and in 50 µM ARL67156, a blocker of NTPDase (red). The slow component is reduced by application of ARL67156, indicating that it is mediated by ATP hydrolysis. Feedback response amplitudes were reduced by ATP application (B), but not by ARL67156 application (C), consistent with the notion that a pH buffer generated by ATP hydrolysis inhibits I_Ca of the cone by keeping the synaptic cleft slightly acidic. (D) Normalized mean feedback responses in six cones in control (pH 7.6; black) and when the superfusate pH was 7.2 (red). The slow component is strongly reduced when the pH gradient between the synaptic and the extrasynaptic compartments is removed. (E–H) The slow component amplitude for individual cells (black) used in (A–D) during control, drug application, and wash. The red line indicates the mean ± sem. In each case, the changes of the slow component amplitude were reversible. The figures also suggest that the slow feedback component may have increased slightly over time. (I) The mean ± sem change in the total feedback amplitude relative to control levels for the results shown in (A–D). Probenecid (<i>p</i> = 0.003), ATP (<i>p</i> = 0.0093), and pH (<i>p</i> = 0.0175) all reduced the feedback amplitude significantly. Only ARL67156 did not change the amplitude significantly (<i>p</i> = 0.2295). (J) The mean ± sem amplitude reduction for the slow component of feedback relative to control values for the results shown in (A–D). In each case, the amplitude reduction of the slow component was significant (probenecid, <i>p</i> = 0.0009; ATP, <i>p</i> = 0.036; ARL67156, <i>p</i> = 0.045; pH, <i>p</i> = 0.010). (K) The change in half activation potentials of I_Ca when 500 µM probenecid, 100 µM ATP, or 50 µM ARL67156 were present in the bath solution or when the extracellular pH was shifted to 7.2. Probenecid did not induce a significant shift of the activation potential of I_Ca (<i>n</i> = 9; <i>p</i> = 0.3375). ATP shifted the half activation potential to more positive potentials (<i>n</i> = 8; <i>p</i> = 0.0277), whereas ARL67156 shifted it to more negative potentials (<i>n</i> = 8; <i>p</i> = 0.0131). Lowering the extracellular pH led to a significant shift of I_Ca to positive potentials (<i>n</i> = 5; <i>p</i> = 0.0022).</p

Feedback consists of a fast and a slow component.

<p>(A) Feedback response measured in a cone in goldfish retina. Cones were clamped at E_Cl (−50 mV) and saturated with a small spot of light. HCs were then hyperpolarized by a 500 ms full-field white light stimulus (4,500 µm). This induced a feedback inward current. The mean amplitude was 11.0±1.1 pA (<i>n</i> = 23). The onset of the feedback response could be fitted best by the sum of two exponentials (red line, sum of the exponential fits; blue lines, individual exponential fits). (B) The amplitude and time constants of the two exponential functions. The fast feedback component (τ_f = 29±3 ms; <i>n</i> = 23) had an amplitude (A_f) that constituted 64±6% of the total feedback response. The remaining 36±6% of the feedback response (A_s) was mediated by a process with a time constant (τ_s) of 189±25 ms (<i>n</i> = 19). (C) Feedback responses in cones from wild-type (WT, black) and Cx55.5^−/− mutant zebrafish (red) were also best fitted by a double exponential function. (D) Compared to goldfish, WT zebrafish had a similar fast component to slow component amplitude ratio (gray: A_f, 68±4%; A_s, 32±4%; <i>n</i> = 13), whereas it shifted in Cx55.5^−/− mutants in favor of the slow feedback component (red: A_f, 52±7%; A_s, 48±7%; <i>n</i> = 9). The amplitude of the slow component of feedback in Cx55.5^−/−mutants (1.62±0.39 pA; <i>n</i> = 9) did not significantly differ from WT (1.87±0.39 pA; <i>n</i> = 13; <i>p</i> = 0.67), showing that the slow component is independent of Cx55.5 hemichannels. Note that the amplitude axes for the WT and mutants are scaled such that it reflects the total reduction of feedback in the mutant relative to WT. The time constant of the fast component (τ_f) did not differ significantly between WT and Cx55.5^−/− mutants (WT, 29.3±3.3 ms; <i>n</i> = 13; Cx55.5^−/−, 40.6±9.6 ms; <i>n</i> = 9; <i>p</i> = 0.21) and was similar to goldfish. The time constant of the slow component (τ_s) in WT and Cx55.5^−/− mutants did not differ significantly (WT, 300.2±52.3 ms; <i>n</i> = 13; Cx55.5^−/−, 408.5±169.7; <i>n</i> = 9; <i>p</i> = 0.49) and was larger than in goldfish.</p

Schematic drawing of the proposed feedback mechanism.

<p>(A) The ephaptic mechanism. Cones continuously release glutamate in the dark via a ribbon (R) synapse optimized for sustained glutamate release. This release depends on the activity of presynaptic Ca²⁺ channels (red). HC dendrites end laterally to the cone synaptic ribbon. Cx hemichannels (green) and Panx1 channels (blue) are expressed on the dendrites of HCs. Because they are both nonspecific channels and are open at the physiological membrane potentials of HCs, a current will flow into the HCs. HC dendrites invaginate the cone synaptic terminal, which leads to a large extracellular resistance (white resistor). The current that flows into HCs via the Cx hemichannels and Panx1 channels has to pass this resistor, which will induce a slight negativity deep in the synaptic cleft. The result will be that the voltage-gated Ca²⁺ channels sense a slightly depolarized membrane potential. When HCs hyperpolarize, the current through the Cx hemichannels will increase and so will the negativity in the synaptic cleft leading to a further decrease of the potential sensed by the cone Ca²⁺ channels. This is the fast component of feedback. (B) The Panx1/ATP-mediated mechanism. Expanded view of the pre- and postsynaptic membranes of cones and HCs. ATP is released by HCs via Panx1 channels. Through a number of steps ATP is converted into inosine, protons, and a phosphate buffer with a pKa of 7.2. This makes the synaptic cleft acidic relative to the extrasynaptic medium (pH 7.6–7.8), which inhibits Ca²⁺ channels and shifts their activation potential to positive potentials. Closing the Panx1 channels prevents HCs from releasing ATP, thereby stopping the production of phosphate buffer, leading to an alkalization of the synaptic cleft. This alkalization disinhibits the Ca²⁺ channels and shifts their activation potential to negative potentials. This mechanism underlies the slow component of feedback. (C) Schematic representation of I_Ca of cones with and without feedback. The black trace shows the I_Ca of cones in the dark. The red trace shows I_Ca when only the ephaptic feedback is active, whereas the blue trace shows the I_Ca when both the ephaptic and Panx1/ATP-mediated feedback are active.</p

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 μm 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