Does Background Matter? A Comparative Characterization of Mouse Models of Autosomal Retinitis Pigmentosa rd1 and Pde6b-KO.

Many retinal degenerative diseases result in vision impairment or permanent blindness due to photoreceptor loss or dysfunction. It has been observed that Pde6brd1 mice (rd1), which carry a spontaneous nonsense mutation in the pde6b gene, have a strong phenotypic similarity to patients suffering from autosomal recessive retinitis pigmentosa. In this study, we present a novel mouse model of retinitis pigmentosa generated through pde6b gene knockout using CRISPR/Cas9 technology. We compare this Pde6b-KO mouse model to the rd1 mouse model to gain insights into the progression of retinal degeneration. The functional assessment of the mouse retina and the tracking of degeneration dynamics were performed using electrophysiological methods, while retinal morphology was analyzed through histology techniques. Interestingly, the Pde6b-KO mouse model demonstrated a higher amplitude of photoresponse than the rd1 model of the same age. At postnatal day 12, the thickness of the photoreceptor layer in both mouse models did not significantly differ from that of control animals; however, by day 15, a substantial reduction was observed. Notably, the decline in the number of photoreceptors in the rd1 model occurred at a significantly faster rate. These findings suggest that the C3H background may play a significant role in the early stages of retinal degeneration.

Adaptation memory in photoreceptors: different mechanisms in rods and cones.

Vertebrate rods and cones operate over a wide range of ambient illumination, which is provided by light adaptation mechanisms regulating the sensitivity and speed of the phototransduction cascade. Three calcium-sensitive feedback loops are well established in both rods and cones: acceleration of the quenching of a light-activated visual pigment and cGMP synthesis by guanylate cyclase, and increased affinity of ion channels for cGMP. Accumulating evidence suggests that the molecular mechanisms of light adaptation are more complex. While investigating these putative mechanisms, we discovered a novel phenomenon, observing that the recovery of light sensitivity in rods after turning off non-saturating adaptive light can take tens of seconds. Moreover, after a formal return of the membrane current to the dark level, cell sensitivity to the stimuli remains decreased for a further 1-2 min. We termed this phenomenon of prolonged photoreceptor desensitization 'adaptation memory' (of previous illumination) and the current study is focused on its detailed investigation in rods and an attempt to find the same phenomenon in cones. In rods, we have explored the dependencies of this phenomenon on adapting conditions, specifically, the intensity and duration of adapting illumination. Additionally, we report that fish and frog red-sensitive cones possess similar features of adaptation memory, such as a drop in sensitivity just after the steps of bright light and slow sensitivity recovery. However, we have found that the rate of this process and its nature are not the same as in rods. Our results indicate that the nature of the temporary drop in the sensitivity in rods and cones after adapting steps of light is different. In the rods, adaptation memory could be attributed to the existence of long-lasting modifications of the components of the phototransduction cascade after adapting illumination. In cones, the observed form of the adaptation memory seems to be due to the sensitivity drop caused by a decrease in the availability of the visual pigment, that is, by bleaching.

Magnetoreceptory Function of European Robin Retina: Electrophysiological and Morphological Non-Homogeneity.

The avian magnetic compass allows orientation during migration and is shown to function properly under short-wavelength but not long-wavelength visible light. Therefore, the magnetoreceptive system is assumed to be light- and wavelength-dependent and localized in the retina of the eye. Putative candidates for the role of primary magnetosensory molecules are the cryptochromes that are known to be expressed in the avian retina and must be able to interact with phototransduction proteins. Previously, we reported that in migratory birds change in magnetic field direction induces significant effects on electroretinogram amplitude in response to blue flashes, and such an effect was observed only in the nasal quadrant of the retina. Here, we report new electroretinographic, microscopic and microspectrophotometric data on European robins, confirming the magnetosensitivity of the retinal nasal quadrant after applying the background illumination. We hypothesized that magnetoreceptive distinction of this region may be related to its morphology and analyzed the retinal distribution and optical properties of oil droplets, the filtering structures within cones. We found that the nasal quadrant contains double cones with the most intensely colorized oil droplets compared to the rest of the retina, which may be related to its magnetosensory function.

Phototransduction in Anuran Green Rods: Origins of Extra-Sensitivity.

Green rods (GRs) represent a unique type of photoreceptor to be found in the retinas of anuran amphibians. These cells harbor a cone-specific blue-sensitive visual pigment but exhibit morphology of the outer segment typical for classic red rods (RRs), which makes them a perspective model object for studying cone-rod transmutation. In the present study, we performed detailed electrophysiological examination of the light sensitivity, response kinetics and parameters of discrete and continuous dark noise in GRs of the two anuran species: cane toad and marsh frog. Our results confirm that anuran GRs are highly specialized nocturnal vision receptors. Moreover, their rate of phototransduction quenching appeared to be about two-times slower than in RRs, which makes them even more efficient single photon detectors. The operating intensity ranges for two rod types widely overlap supposedly allowing amphibians to discriminate colors in the scotopic region. Unexpectedly for typical cone pigments but in line with some previous reports, the spontaneous isomerization rate of the GR visual pigment was found to be the same as for rhodopsin of RRs. Thus, our results expand the knowledge on anuran GRs and show that these are even more specialized single photon catchers than RRs, which allows us to assign them a status of "super-rods".

Electroretinographic study of the magnetic compass in European robins.

Migratory birds are known to be sensitive to external magnetic field (MF). Much indirect evidence suggests that the avian magnetic compass is localized in the retina. Previously, we showed that changes in the MF direction could modulate retinal responses in pigeons. In the present study, we performed similar experiments using the traditional model animal to study the magnetic compass, European robins. The photoresponses of isolated retina were recorded using ex vivo electroretinography (ERG). Blue- and red-light stimuli were applied under an MF with the natural intensity and two MF directions, when the angle between the plane of the retina and the field lines was 0° and 90°, respectively. The results were separately analysed for four quadrants of the retina. A comparison of the amplitudes of the a- and b-waves of the ERG responses to blue stimuli under the two MF directions revealed a small but significant difference in a- but not b-waves, and in only one (nasal) quadrant of the retina. The amplitudes of both the a- and b-waves of the ERG responses to red stimuli did not show significant effects of the MF direction. Thus, changes in the external MF modulate the European robin retinal responses to blue flashes, but not to red flashes. This result is in a good agreement with behavioural data showing the successful orientation of birds in an MF under blue, but not under red illumination.

Searching for magnetic compass mechanism in pigeon retinal photoreceptors.

Migratory birds can detect the direction of the Earth's magnetic field using the magnetic compass sense. However, the sensory basis of the magnetic compass still remains a puzzle. A large body of indirect evidence suggests that magnetic compass in birds is localized in the retina. To confirm this point, an evidence of visual signals modulation by magnetic field (MF) should be obtained. In a previous study we showed that MF inclination impacts the amplitude of ex vivo electroretinogram (ERG) recorded from isolated pigeon retina. Here we present the results of an analysis of putative MF effect on one component of ERG, the photoreceptor's response, isolated from the total ERG by adding sodium aspartate and barium chloride to the perfusion solution. Photoresponses were recorded from isolated retinae of domestic pigeons Columba livia. The retinal samples were placed in MF that was modulated by three pairs of orthogonal Helmholtz coils. Light stimuli (blue and red) were applied under two inclinations of MF, 0° and 90°. In all the experiments, preparations from two parts of retina were used, red field (with dominant red-sensitive cones) and yellow field (with relatively uniform distribution of cone color types). In contrast to the whole retinal ERG, we did not observe any effect of MF inclination on either amplitude or kinetics of pharmacologically isolated photoreceptor responses to blue or red half-saturating flashes. A possible explanations of these results could be that magnetic compass sense is localized in retinal cells other than photoreceptors, or that photoreceptors do participate in magnetoreception, but require some processing of compass information in other retinal layers, so that only whole retina signal can reflect the response to changing MF.

Rejection of the biophoton hypothesis on the origin of photoreceptor dark noise.

Rod photoreceptors of the vertebrate retina produce, in darkness, spontaneous discrete current waves virtually identical to responses to single photons. The waves comprise an irreducible source of noise (discrete dark noise) that may limit the threshold sensitivity of vision. The waves obviously originate from acts of random activation of single rhodopsin molecules. Until recently, it was generally accepted that the activation occurs due to the rhodopsin thermal motion. Yet, a few years ago it was proposed that rhodopsin molecules are activated not by heat but rather by real photons generated within the retina by chemiluminescence. Using a high-sensitive photomultiplier, we measured intensities of biophoton emission from isolated retinas and eyecups of frogs (Rana ridibunda) and fish (sterlet, Acipenser ruthenus). Retinal samples were placed in a perfusion chamber and emitted photons collected by a high-aperture quartz lens. The collected light was sent to the photomultiplier cathode through a rotating chopper so that a long-lasting synchronous accumulation of the light signal was possible. The absolute intensity of bio-emission was estimated by the response of the measuring system to a calibrated light source. The intensity of the source, in turn, was quantified by measuring rhodopsin bleaching with single-rod microspectrophotometry. We also measured the frequency of discrete dark waves in rods of the two species with suction pipette recordings. Expressed as the rate constant of rhodopsin activation, it was 1.2 × 10-11/s in frogs and 7.6 × 10-11/s in sterlets. Approximately two thirds of retinal samples of each species produced reliably measurable biophoton emissions. However, its intensity was ≥100 times lower than necessary to produce the discrete dark noise. We argue that this is just a lower estimate of the discrepancy between the hypothesis and experiment. We conclude that the biophoton hypothesis on the origin of discrete dark noise in photoreceptors must be rejected.

Developmental prefrontal mRNA expression of D2 dopamine receptor splice variants and working memory impairments in rats after early life Interleukin-1ß elevation.

Long (D2L) and Short (D2S) isoforms of D2 dopamine receptor differ in their biochemical and physiological properties, which could affect functioning of prefrontal cortex. Contribution of distinct D2 dopamine receptor isoforms to cognitive dysfunctions and its developmental regulation are currently not fully elucidated. In the present study, we evaluated developmental mRNA expression of D2S/D2L dopamine receptor isoforms within the rat medial prefrontal cortex (mPFC) in the model of neurodevelopmental cognitive dysfunction. Working memory performance (Y-maze spontaneous alternations) and D2S/D2L mRNA expression in the mPFC (by qRT-PCR) were evaluated in juvenile (P27), adolescent (P42-47) and adult (P75-90) rats after chronic early life treatment with proinflammatory cytokine interleukin (IL)-1ß (1 µg/kg i.p. daily P15-21). It was shown that IL-1ß elevation during the 3rd week of life leads to working memory deficit originating in juvenile animals and persisting into adulthood. D2S mRNA expression was strongly downregulated during adolescence, and such downregulation was exaggerated in animals injected with IL-1ß during P15-21. Early life IL-1ß administrations influenced developmental changes in the D2S/D2L mRNA ratio. This measure was found to be decreased in adolescent and adult control (intact and vehicle-treated) rats compared to juvenile control, while in the case of IL-1ß-treated animals, the decrease in D2S/D2L ratio was observed only in adulthood but not in adolescence compared to juvenile rats. During the adolescence, D2S mRNA expression was downregulated and D2S/D2L ratio was upregulated in the mPFC of rats treated with IL-1ß during the 3rd week of life compared to controls. Based on these data we conclude that changes in the developmental expression of D2 dopamine receptor splice variants within mPFC may underlie long-lasting cognitive deficit associated with neonatal pathology.

Origins of the phototransduction delay as inferred from stochastic and deterministic simulation of the amplification cascade.

PURPOSE: To identify steps of the phototransduction cascade responsible for the delay of the photoresponse. METHODS: Electrical responses of fish (Carassius) cones and Rana ridibunda frog rods and cones were recorded with a suction pipette technique and as an aspartate-isolated mass receptor potential from isolated perfused retinas. Special attention was paid to sufficiently high temporal resolution (1-ms flash, 700 Hz amplification bandpass). Stochastic simulation of the activation steps from photon absorption to the formation of catalytically active phosphodiesterase (PDE) was performed. In addition, a deterministic mathematical model was fit to the experimental responses. The model included a detailed description of the activation steps of the cascade that enabled identification of the role of individual transduction stages in shaping the initial part of the response. RESULTS: We found that the apparent delay of the photoresponse gets shorter with increasing stimulus intensity and reaches an asymptotic value of approximately 3 ms in cones and greater than or equal to 10 ms in rods. The result seems paradoxical since it is suggested that the delay occurs in the chain of steps from photon absorption to the formation of active transducin (T*) which in cones is, on average, slower than in rods. Stochastic simulation shows that actually the steps from photon absorption to T* may not contribute perceptibly to the delay. Instead, the delay occurs at the stage that couples the cycle of repetitive activation of T by rhodopsin (R*) with the activation of PDE. These steps include formation of T* (= T α GTP) out of T αßγ GTP released from the activation cycle and the subsequent interaction of T* with PDE. This poses a problem. The duration of an average cycle of activation of T in rods is approximately 5 ms and is determined by the frequency of collisions between R* and T in the photoreceptor membrane. The frequency is roughly proportional to the surface packing density of T in the membrane. As the packing density of PDE is approximately 12 times lower than that of T, it could be expected that the rate of the T*-PDE interaction were an order of magnitude slower than that of R* and T. As modeling shows, this is the case in rods. However, the delay in cones is approximately 3 ms which could be achieved only at a T*-PDE interaction time of less than or equal to 5 ms. This means that either the frequency of the collisions of T* and PDE, or the efficiency of collisions, or both in cones are approximately ten times higher than in rods. This may be a challenge to the present model of the molecular organization of the photoreceptor membrane. CONCLUSIONS: The delay of the photoresponse is mainly set by the rate of interaction of T* with PDE. In cones, the delay is shorter than in rods and, moreover, shorter than the duration of the cycle of repetitive activation of T by R*. This poses a problem for the present model of diffusion interaction of phototransduction proteins in the photoreceptor membrane.