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.
Date of Award | 2003 |
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Original language | English |
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Awarding Institution | |
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AN INVESTIGATION OF THE PHOTOTRANSDUCTION CASCADE AND TEMPORAL CHARACTERISTICS OF THE RETINA OF THE CUTTLEFISH, SEPIA OFFICINALIS
NELSON, L. (Author). 2003
Student thesis: PhD