AN INVESTIGATION OF THE PHOTOTRANSDUCTION CASCADE AND TEMPORAL CHARACTERISTICS OF THE RETINA OF THE CUTTLEFISH, SEPIA OFFICINALIS
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Cephalopods have extremely well developed visual systems which are of particular interest due to the well known morphological similarity of the cephalopod eye to the vertebrate eye. This similarity ends at the level of the photoreceptors where vertebrates and invertebrates have been found to use different intracellular second messengers. Although the effect of extracellular ion manipulation on the light response has been examined and some very useful biochemical studies carried out, the pathway has not been investigated by the use of pharmacological intervention; a method which has proved to be useful in other preparations. This study examines various properties of the photoreceptors of the cuttlefish, Sepia officinalis, with particular interest in the second messenger signalling pathway. Both extracellular and whole cell patch clamp recording has been utilised. The second messenger signalling pathway, which mediates phototransduction in the retina of S. officinalis, was investigated by recording the electroretinogram and examining how this changed with the application of various extracellularly applied, membrane permeable pharmacological agents. Invertebrate phototransduction utilises the phosphoinositide (PI) signalling pathway therefore specific activators and inhibitors targeted at precise sites of this pathway were applied to the extracellular bathing solution. These studies indicated that cleavage of phosphatidylinositol-4,5-bisphosphate is essential for the production of a light response and that the inositol trisphophate (IP3) branch of this pathway is of greatest importance in this preparation, as opposed to the diacylglycerol branch. How this second messenger cascade transfers the incoming information into a temporally coded signal was studied by measuring maximum critical flicker fusion frequency. The effect of cell size on this property was investigated and also how cell sensitivity was affected and whether these properties appeared to fit the animal's environmental conditions or whether they were restricted by cellular properties. The animals were found to have relatively "slow" eyes. However the younger age group studied, with shorter photoreceptors, was found to be both faster and more sensitive. This was an unexpected finding considering temporal resolving power is often sacrificed for sensitivity. It is suggested that the observed differences between age groups was attributable to the effects of increased cell size on the cell membrane time constant and that deterioration of signalling molecules with aging may also be a contributing factor. An investigation of the cell signalling pathway at the level of individual cells was also carried out using the whole cell patch clamp technique. Using this technique, two voltage activated currents were found; an inward sodium current characterised by its voltage and tetrodotoxin sensitivity, and an outward potassium current characterised by its tetraethylammonium sensitivity. As well as finding further evidence for the involvement of the IP3 branch of the PI pathway there is also evidence of a role for cyclic guanosine monophosphate. A suitable mode of measuring light-induced fluctuations in the intracellular calcium levels was also investigated with a view to observing the impact of the pharmacological agents on intracellular calcium concentration. This investigation has enhanced the understanding of the S. officinalis visual system by greatly adding to the present knowledge of the second messenger signalling cascade and by giving an insight into how this transfers into the animal's temporal resolving power. Some preliminary information regarding the membrane currents activated by light has also been presented. This has all been possible by the development of a versatile retinal slice preparation that has been proven to be accessible to extracellular recording and whole cell patch clamp recording combined with pharmacological manipulation.
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