Periodic Reporting for period 1 - COLOURFUL DARKNESS (Colour vision in the dark: ecological, physiological and structural basis of rod-rod colour opponency in the frog retina)
Berichtszeitraum: 2021-05-01 bis 2023-04-30
Most vertebrates, including humans, cannot ‘see colour in the dark’ because the retinal rod photoreceptors that mediate vision in dim light usually come in a single spectral flavour (“green”), precluding spectral comparisons. But frogs and some salamanders have an additional ”blue”-rod type, potentially allowing for purely rod-based colour discrimination. Using behaviour, I recently demonstrated that this is indeed the case: frogs do make spectral comparisons down to the absolute visual sensitivity threshold. However, the ecological relevance of nocturnal colour vision and the structure and function of the retinal circuitry underlying rod-rod spectral comparisons, and enabling the simultaneous preservation of sensitivity and spectral resolution, have not been explored.
Thus, this proposal sought to establish the purpose as well as the underlying retinal physiology and circuit implementation of frog colour discrimination near the visual threshold. For this, we proposed to combine natural scene imaging, high-throughput multi-electrode array recordings from 1,000s of retinal ganglion cells and synaptic resolution serial section electron microscopy of the blue-sensitive rod circuitry.
This would enable unravelling which visual information is available to be used, and how it is processed, to reach the low-light limits of colour discrimination performance available to the vertebrate eye. It would also provide a link between the more intensely studied retinal circuits of fish and mammals, providing important insights about the evolution of vertebrate retinal networks’ architecture and computations. Finally, the topic of ‘colour vision in the dark’ is one that attracts considerable public interest, so this project would create valuable opportunities for public engagement with the research supported by the European Commission.
From the results obtained so far, we can conclude that in dim illumination frog retinas use a computational strategy for encoding colour that is different from the ‘classical’ colour discrimination mechanism, with input from the blue-sensitive rods apparently having a suppressive role over input from the green-sensitive rods. From the electron microscopy dataset we have also been able to conclude that the internal anatomy of the blue-sensitive rod differs between frog species.
Thus, this proposal sought to establish the purpose as well as the underlying retinal physiology and circuit implementation of frog colour discrimination near the visual threshold. For this, we proposed to combine natural scene imaging, high-throughput multi-electrode array recordings from 1,000s of retinal ganglion cells and synaptic resolution serial section electron microscopy of the blue-sensitive rod circuitry.
This would enable unravelling which visual information is available to be used, and how it is processed, to reach the low-light limits of colour discrimination performance available to the vertebrate eye. It would also provide a link between the more intensely studied retinal circuits of fish and mammals, providing important insights about the evolution of vertebrate retinal networks’ architecture and computations. Finally, the topic of ‘colour vision in the dark’ is one that attracts considerable public interest, so this project would create valuable opportunities for public engagement with the research supported by the European Commission.
From the results obtained so far, we can conclude that in dim illumination frog retinas use a computational strategy for encoding colour that is different from the ‘classical’ colour discrimination mechanism, with input from the blue-sensitive rods apparently having a suppressive role over input from the green-sensitive rods. From the electron microscopy dataset we have also been able to conclude that the internal anatomy of the blue-sensitive rod differs between frog species.
We have investigated retinal responses to different flashes of light with a multi-electro array (MEA) setup for electrophysiological recordings. Responses are more pronounced in the green part of the visible spectrum when the flashes were dim, and in the red part of the spectrum when the flashes were bright. This matches previous knowledge about frog retinas being more sensitive to red in bright light, and more sensitive to green in dim light. Overall, the retinas showed higher activity when the light flashes went off than when they went on, regardless of the colour or the intensity of the flash. However, for blue flashes in particular the activity was lower when dimmer flashes were used compared to the activity when brighter flashes were used. The activity when the flashes went off was the same in all cases. This response pattern represents a potential mechanism for encoding colour information, and we are currently investigating it in further detail using a more comprehensive battery of different types of flashes.
We have also studied the anatomy of the cells in the retina and their connections, at a magnification high enough to identify synaptic contacts between cells. For this we got a dataset composed of more than 17,000 electron microscopy images covering around 1,900 slices of tissue, with a total volume of 0.1x0.1x0.1 mm. We used an image processing software to trace individual cells across all the slices of the sample and discovered that the blue-sensitive rods from the frogs that we are using have a different microanatomy than those of other frog species; in particular with regards to the distribution of mitochondria within the cell. We are now putting together the rest of the puzzle by tracing the cells that connect to the blue-sensitive rods, and in turn following them to identify which other cells they connect to.
All this work done so far has been or will be shortly presented at scientific conferences, and the presentations are openly available in the lab website: https://badenlab.org/posters/(öffnet in neuem Fenster). Once we finish with the ongoing experiments and measurements the whole work will be published as an article in a scientific journal and will also be openly available.
Finally, we have done some extra work involving tadpoles instead of adult frogs. We teamed with another research group to generate tadpoles in which the neurons become fluorescent when they are active, such that they can be identified and their activity measured using a 2-photon microscope. We have been able to obtain tadpoles with fluorescent signals strong enough for this purpose, and will continue using them to study the development of the visual system.
We have also studied the anatomy of the cells in the retina and their connections, at a magnification high enough to identify synaptic contacts between cells. For this we got a dataset composed of more than 17,000 electron microscopy images covering around 1,900 slices of tissue, with a total volume of 0.1x0.1x0.1 mm. We used an image processing software to trace individual cells across all the slices of the sample and discovered that the blue-sensitive rods from the frogs that we are using have a different microanatomy than those of other frog species; in particular with regards to the distribution of mitochondria within the cell. We are now putting together the rest of the puzzle by tracing the cells that connect to the blue-sensitive rods, and in turn following them to identify which other cells they connect to.
All this work done so far has been or will be shortly presented at scientific conferences, and the presentations are openly available in the lab website: https://badenlab.org/posters/(öffnet in neuem Fenster). Once we finish with the ongoing experiments and measurements the whole work will be published as an article in a scientific journal and will also be openly available.
Finally, we have done some extra work involving tadpoles instead of adult frogs. We teamed with another research group to generate tadpoles in which the neurons become fluorescent when they are active, such that they can be identified and their activity measured using a 2-photon microscope. We have been able to obtain tadpoles with fluorescent signals strong enough for this purpose, and will continue using them to study the development of the visual system.
Our main finding so far from the electrophysiological recordings is the discovery of a potential colour-processing strategy underlying colour discrimination which doesn’t rely on the classical paradigm. This result adds to mounting evidence about how the same basic retinal architecture shared by all vertebrates can give raise to several different ways (unheard of until recently) in which colour information can be processed and used.
From the anatomical point of view, what was initially the main obstacle for the progress of the tracings ended up being our main finding working with the electron microscopy dataset so far: the unveiling of different morphological features in Xenopus blue-sensitive rods compared to what was known for other amphibians.
Our progress so far also highlights the importance of sustained research using different animal models to unveil fundamental mechanisms and anatomical bases of information processing by sensory systems, which are crucial for understanding the limitations imposed by aging, diseases and developmental disorders, and the potential to develop therapeutic tools using alternative pathways.
From the anatomical point of view, what was initially the main obstacle for the progress of the tracings ended up being our main finding working with the electron microscopy dataset so far: the unveiling of different morphological features in Xenopus blue-sensitive rods compared to what was known for other amphibians.
Our progress so far also highlights the importance of sustained research using different animal models to unveil fundamental mechanisms and anatomical bases of information processing by sensory systems, which are crucial for understanding the limitations imposed by aging, diseases and developmental disorders, and the potential to develop therapeutic tools using alternative pathways.