Neuropeptide Y (NPY)-like immunoreactive amacrine cells in retinas of frog and goldfish

Summary The distribution of neuropeptide Y (NPY)-like immunoreactivity in rat, rabbit, chick, frog and goldfish retinas was investigated by immunohistochemistry. Positive results were observed only in the frog and goldfish retinas. NPY immunoreactivity was associated with a small population of amacrine cell bodies in the inner nuclear layer and cell processes in the inner plexiform layer of both retinas. In the frog retina, three distinct layers containing immunoreactivity were observed in the inner plexiform layer. In contrast, the immunoreactivity in the same area of the goldfish retina was more or less separated into two layers. Convincing evidence could not be found for the co-existence of NPY-like material with other putative transmitter-like substances in the two retinas.Radioimmunoassay revealed the presence of small amounts of NPY-like immunoreactivity in the rabbit retina; the goldfish and frog retinas contained significantly more immunoreactive material. High performance liquid chromatography of the immunoreactive material in frog and goldfish retinas showed each retina containing different molecular forms of NPY-like proteins, neither of which resembled porcine NPY or PYY.The endogenous NPY-like material of the frog retina can be released by potassium depolarisation in a calciumdependent way. In view of all these data an NPY-like protein must now be considered a potential retinal transmitter.     

Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long-term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore-fibroblast co-culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast-derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane-enclosed melanosomes in a process that was upregulated by alpha-melanocyte-stimulating hormone (alpha-MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane-proteins seemed to be of importance, as the cluster-like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long-term colour changes and for studies of factors that affect pigmentation.     

Dll1 and Dll4 function sequentially in the retina and pV2 domain of the spinal cord to regulate neurogenesis and create cell diversity

Signalling mediated by Notch receptors is known to have multiple functions during vertebrate neural development, regulating processes like progenitor differentiation and cell type diversification. Various Notch ligands are expressed in the developing nervous system and their activities might contribute to this multiplicity of functions. Here, we show that two Delta-like genes, Dll1 and Dll4, are sequentially expressed in differentiating neurons of the embryonic mouse retina and spinal cord's pV2 domain, with Dll1 starting to be expressed before Dll4. Analysis of Dll1 mutants reveals this gene is necessary and sufficient to maintain a pool of progenitors in the embryonic neuroepithelium. Accordingly, in the spinal cord domains where Dll1 is the only expressed Notch ligand, its inactivation leads to an increased rate of neurogenesis and premature differentiation of neural progenitors. In contrast, in the pV2 domain and retina where Dll1 is co-expressed with Dll4, progenitors are not exhausted and cell diversity is maintained. Together, our results support a model where Dll1 and Dll4 are part of a unique genetic circuitry that regulates subsequent steps of neurogenesis in the retina and pV2 domain: while Dll1 serves to prevent the untimely differentiation of neural progenitors, Dll4 might function to generate diversity within the population of differentiating neurons.     

Pharmacological Properties of Glycine Transport in the Frog Retina

The high-affinity glycine transport in neurons and glial cells is the primary means for inactivating synaptic glycine. Two different glycine transporter genes, Glyt-1 and Glyt-2, have been cloned. Glyt-1 has been reported to occur in the retina, but there is no evidence for expression of the Glyt-2 transporter. We have pharmacologically characterized glycine transport in the frog retina. 3H-Glycine uptake in the retina was insensitive to modulation by phorbol esters or changes in cAMP levels, and was partially inhibited by sarcosine. Differential sensitivity of glycine transport to sarcosine was exhibited by synaptosomal fractions from the inner and outer plexiform layers of the frog retina. The Na+ Hill coefficient of glycine uptake was 2.0, as has been reported for Glyt-2. In addition, amoxapine, a specific inhibitor of the Glyt-2a isoform, reduced by 60% glycine uptake by P2 synaptosomal fraction. Our results indicate the presence of different glycine transporter isoforms in the frog retina, acting mainly through the classical inhibitory glycine system.     

Dark adaptation of the photoreceptors in the isolated frog retina during induced lipid peroxidation

The influence of lipid peroxidation (LPO) in isolated frog retina on dark adaptation of photoreceptors was studied. Stimulus-response functions, late receptor potential (LRP) as function of the stimulus light intensity were measured before bleach and in a steady state after dark adaptation. It was shown that accumulation of LPO products influenced dark adaptation in photoreceptors. Based on the displacements of the stimulus-response curves and experimental measurement data on the rate of LRP collapse after retina treatment with strophanthin it is concluded that the most probable mechanism of such an influence lies in a change of photoreceptor plasma membrane permeability.     

Adaptive Colour Contrast Coding in the Salamander Retina Efficiently Matches Natural Scene Statistics

The visual system continually adjusts its sensitivity to the statistical properties of the environment through an adaptation process that starts in the retina. Colour perception and processing is commonly thought to occur mainly in high visual areas, and indeed most evidence for chromatic colour contrast adaptation comes from cortical studies. We show that colour contrast adaptation starts in the retina where ganglion cells adjust their responses to the spectral properties of the environment. We demonstrate that the ganglion cells match their responses to red-blue stimulus combinations according to the relative contrast of each of the input channels by rotating their functional response properties in colour space. Using measurements of the chromatic statistics of natural environments, we show that the retina balances inputs from the two (red and blue) stimulated colour channels, as would be expected from theoretical optimal behaviour. Our results suggest that colour is encoded in the retina based on the efficient processing of spectral information that matches spectral combinations in natural scenes on the colour processing level.     

Behavioural,ecological, and evolutionary aspects of diversity in frog colour patterns

The role of colours and colour patterns in behavioural ecology has been extensively studied in a variety of contexts and taxa, while almost overlooked in many others. For decades anurans have been the focus of research on acoustic signalling due to the prominence of vocalisations in their communication. Much less attention has been paid to the enormous diversity of colours, colour patterns, and other types of putative visual signals exhibited by frogs. With the exception of some anecdotal observations and studies, the link between colour patterns and the behavioural and evolutionary ecology of anurans had not been addressed until approximately two decades ago. Since then, there has been ever‐increasing interest in studying how colouration is tied to different aspects of frog behaviour, ecology and evolution. Here I review the literature on three different contexts in which frog colouration has been recently studied: predator–prey interactions, intraspecific communication, and habitat use; and I highlight those aspects that make frogs an excellent, yet understudied, group to examine the role of colour in the evolution of anti‐predation strategies and animal communication systems. Further, I argue that in addition to natural‐history observations, more experiments are needed in order to elucidate the functions of anuran colouration and the selective pressures involved in its diversity. To conclude, I encourage researchers to strengthen current experimental approaches, and suggest future directions that may broaden our current understanding of the adaptive value of anuran colour pattern diversity.     

Microscopic investigation of the comparative morphology of the retina

Morphological differences in the architectonics (the relations and composition of the layers and sublayers) of the retina are described in various vertebrates: pike, frog, and cat. These differences apply to both cellular and plexiform layers. The differences are particularly marked in the composition of the sublayers of the inner nuclear layer. In the frog the greatest degree of subdivision into layers of processes of the ganglion and amacrine cells is observed to correspond to the particularly complex differentiation of the inner plexiform layer of the retina (about 10 sublayers). In all the animals studied the ganglion cells can be divided into two principal types: symmetrical and asymmetrical, with many varieties. Asymmetrical amacrine cells are found in the pike and frog retina. The presence of vertical processes branching in the outer plexiform layer is confirmed for amacrine cells in the cat retina. The structural features of the retina are discussed in connection with physiological findings.     

Biosynthesis of Somatostatin-Like Immunoreactivity by Frog Retinas In Vitro

Abstract: Somatostatin-like immunoreactivity has been localized to a wide variety of central nervous system neurons, including the retina. We utilized the unique advantages the retina provides for in vitro studies of nerves to examine the biosynthesis of somatostatin. Extracts of frog retinas pulse-labeled with [³⁵S]cysteine for various time periods revealed uptake of radioactivity into material adsorbable by anti-somatostatin antibody linked to affinity beads. This uptake increased in a curvilinear fashion for 4 h and was inhibited by cycloheximide (0.2 m M ) or by boiling the retinas prior to labeling. Pulse-chase experiments revealed that affinity-adsorbable radioactivity from retinal extracts decreased with time of incubation in chase medium; 89% of this decrease could be accounted for by increases in the affinity-adsorbable radioactivity of the chase medium. Chromatography of the retinal extracts on Sephadex G50 (superfine) revealed four elution peaks, whereas only one peak, co-eluted with somatostatin-14, could be identified in the medium. Chromatographic elution patterns of affinity-adsorbable radioactivity from extracts of pulse-labeled retinas incubated in chase medium for various times showed a gradual shift of radioactivity from the earlier-eluting peaks to the later ones. These studies indicate that biosynthesis of somatostatin occurs in frog retinas in vitro. The retina may be a useful model for further study of peptidergic neurons.     

Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo1

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long‐term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore‐fibroblast co‐culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast‐derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane‐enclosed melanosomes in a process that was upregulated by α‐melanocyte‐stimulating hormone (α‐MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane‐proteins seemed to be of importance, as the cluster‐like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long‐term colour changes and for studies of factors that affect pigmentation.     

Cast adrift on an island: introduced populations experience an altered balance between selection and drift

A long-standing question in evolutionary biology is what becomes of adaptive traits when a species expands its range into novel environments. Here, we report the results of a study on an adaptive colour pattern polymorphism (stripes) of the coqui frog, Eleutherodactylus coqui, following its introduction to Hawaii from Puerto Rico. We compared population differentiation ( ) for the stripes locus-which underlies this colour pattern polymorphism-with neutral microsatellite loci to test for a signature of selection among native and introduced populations. Among native populations, for stripes were lower than expected under the neutral model, suggesting uniform balancing selection. Alternatively, among introduced populations, for stripes did not differ from the neutral model. These results suggest that the evolutionary dynamics of this previously adaptive trait have become dominated by random genetic drift following the range expansion.     

Guanosine triphosphate complex in rod outer segments of the frog retina

It is shown that nearly 70% water--soluble protein of the frog retina outer segments (ROS) consist of three polypeptides with molecular weights 39 000, 36 000 and less than 15 000 daltons. These proteins are present in equal proportions and are, apparently, the subunits of a tightly bound protein complex. The subunit of 39 000 daltons is responsible for guanyl nucleotides binding. Parameters of the investigated GTP-binding complex are similar to transducyn which transmits excitation from bleached rhodopsin to PDE molecules in the bovine retina ROS. The thermodynamic state of GTP-binding protein in frog retina ROS depends on the functional state of the photoreceptor membrane, as shown by microcalorimetric method.     

Predator response to the coloured eyespots and defensive posture of Colombian four-eyed frogs

Deimatic displays, where sudden changes in prey appearance elicit aversive predator reactions, have been suggested to occur in many taxa. These (often only putative) displays frequently involve different components that may also serve antipredator functions via other mechanisms (e.g., mimicry, warning signalling, body inflation). The Colombian four-eyed frog, Pleurodema brachyops, has been suggested to gain protection against predation through putative deimatic displays where they inflate and elevate the posterior part of their body revealing eye-like colour markings. We exposed stationary artificial frogs to wild predators to test whether the two components (eyespot/colour markings, defensive posture) of their putative deimatic display, and their combination, provide protection from predation without the sudden change in appearance. We did not detect any obvious additive effect of defensive posture and eyespots/colour markings on predation risk, but found a marginally significant trend for model frogs in the resting posture to be less attacked when displaying eyespots/colour markings than when they were not, suggesting that the presence of colour markings/eyespots may provide some protection on its own. Additionally, we found that models in a resting posture were overall more frequently attacked on the head than models in a defensive posture, indicating that a defensive posture alone could help redirect predator attacks to non-vital parts of the body. The trends found in our study suggest that the different components of P. brachyops' coloration may serve different functions during a deimatic display, but further research is needed to elucidate the role of each component when accompanied by sudden prey movement.     

Identification and characterization of protein kinase C-related enzymes in frog retina

We isolated two types of protein kinase C-related enzymes (fPKC) in frog (Rana catesbeiana) retina. Three sequential steps of column chromatography of DEAE-cellulose, butyl-Toyopearl, and hydroxylapatite were carried out and resulted in the separation of two types of distinct Ca2+/phospholipid-dependent protein kinase activities. These proteins reacted with antibodies raised against synthetic peptides based on the predicted amino acid sequences of rat protein kinase C delta or zeta subspecies, and showed molecular masses of 90 and 75 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Immunocytochemical analysis of 90 and 75K fPKC in frog retina revealed that the 75K fPKC was localized in the outer segment of cone cells and in the microvillar projections of Müller cells. These results suggested that fPKCs play differential roles on the photosignal transduction in frog retina.     

Comparative studies on the effects of some enkephalin agonists on the ERG of frog and turtle

1. Effects of mu-agonist morphine in a concentration of 1.10(-5)-1.10(-6) M and delta-agonist Dalargin in a concentration of 1.10(-5)-1.10(-8) M on electroretinogram (ERG) of frog (mixed type of retina) and turtle (predominantly cone retina) were investigated. 2. The enkephalin agonists studied influenced both types of retina in a different manner, producing mainly inhibitory effects. 3. Variability in mu-receptors sensitivity in the turtle retina, most probably depending on the seasons, was observed. 4. The data obtained show that opiate-sensitive mechanisms control the bioelectric activity of the distal layers of frog and turtle retina. 5. Species specificity and variability in sensitivity of the opiate receptors are discussed.     

Formation of hypsorhodopsin in frog retina

Hypsorhodopsin was formed in frog retina by irradiation at liquid helium temperature and converted into bathorhodopsin above about 29 K.     

Formation of hyposorhodopsin in frog retina.

Hypsorhodopsin was formed in frog retina by irradiation at liquid helium temperature and converted into bathorhodopsin above about 29K.     

The butterfly Papilio xuthus detects visual motion using chromatic contrast

Many insects’ motion vision is achromatic and thus dependent on brightness rather than on colour contrast. We investigate whether this is true of the butterfly Papilio xuthus, an animal noted for its complex retinal organization, by measuring head movements of restrained animals in response to moving two-colour patterns. Responses were never eliminated across a range of relative colour intensities, indicating that motion can be detected through chromatic contrast in the absence of luminance contrast. Furthermore, we identify an interaction between colour and contrast polarity in sensitivity to achromatic patterns, suggesting that ON and OFF contrasts are processed by two channels with different spectral sensitivities. We propose a model of the motion detection process in the retina/lamina based on these observations.