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Radiation is typically of two types, short wave radiation and long wave radiation. Long wave radiation is also known as infrared radiation and is from the earth and sources near it. Visible light, a form of short wave radiation, is one of the most familiar form of electromagnetic radiation. It is detected by the human eye in a variety of colours from red all the way through to green, blue, violet and indigo otherwise known as the visible spectrum. Visible spectrum is part of the much broader spectrum; the electromagnetic spectrum. This can be further divided into boundaries, firstly, wavelength from cosmic rays (short waves) to electromagnetic waves (long waves). Length of waves range from as small as microscopic to large waves of kilometres. Secondly, frequency, which is related to wavelength. Long wave have a low frequency whereas short waves have higher frequencies. Finally photon energy (joules or watt seconds) is low in long wave and high in short waves. Only a certain length of the electromagnetic spectrum is detected by our vision system. These visible wave lengths consist between 400-700 nanometres (nm).
Light is composed of particle-like packets of energy known as photons [Sherwood., 2008]. Wave length on the electromagnetic spectrum range from 10â»4 m to 104 [Sherwood., 2008]. Light of different wavelengths correlated as different sensation of colour in this particular small section of the electromagnetic spectrum. The colour blue and violet are perceived at short wavelengths and orange and red are seen at longer wavelengths. Light rays travel in a straight line until they interact with molecules and atoms of the atmosphere and objects around us. The light rays can bounce off the surface depending on the angle of which the light strikes, this is known as reflection. They can be absorbed as described earlier and also the light rays can form images in the eye by refraction. Refraction is when the light rays bend and travel from one transparent medium to another [Bear., 2007]
Anatomy of the eye
The eye is composed of ectoderm which is the outer layer of one of three germ cell layers of the early embryo and mesenchymal which is part of the embryonic mesoderm which goes onto become connective tissue, bone, cartilage and much more. The ectoderm goes onto develop the retina, nerve fibres of the optic nerve and smooth muscle of the iris. Head side surface of the ectoderm forms the lens, coneal and conjunctival epithelium, and the lacrimal and tarsal glands. Mesenchyme goes onto form the corneal stroma, the sclera, the charioid, the iris, the cilliary musculature, part of the vitreous body and the cells lining the anterior chamber [Snell and Lemp., 1998]
The structure of the eye can be interrupted in three ways; its gross anatomy, ophthalmoscopic appearance and the cross-sectional anatomy. Firstly looking at the gross anatomy, when looking into a person's eye the main structure visible are the pupil which allows light to enter and travel to the retina. The pupil appears dark due to the light absorbing pigments in the retina. Surrounding the pupil is the iris which determines eye colour due to its pigmentation. The iris is also made up of two muscles which help to vary the size of the pupil. One muscle causes the pupil to become smaller when it is contracting and the other muscle allows the iris to become larger. The cornea a transparent external surface covers both the pupil and iris. The cornea continues with the sclera which is the white of the eye. The eyeball rests inside the eye orbit which is a bony socket of the skull. The sclera is connected to three pairs of extraocular muscle which allows movement of the eye in the orbit. Conjunctiva covers the muscle therefore is not visible. The optic nerve at the back of the eye carries axons from the retina to part of the brain near the pituitary gland.
Ophthalmoscopic appearance of the eye is assisted by the ophthalmoscope. The ophthalmoscope allows to see through the pupil to the retina. Main feature visible are the blood vessels on the surface of the retina. These retinal vessels originate from the optic disk were the optic nerve fibres exit the retina [Bear et al., 2007]. Light sensation is not observed at the optic disk as there are no photoreceptors present. And this also occurs were large blood vessels exit as they cast a shadow on the retina thus blocking the light. In the centre of the retina a darker coloured region known as the macula which allows central vision. Besides the colour of the macula appearing darker then the retina with a yellowish hue, it can also be recognised by the absence of large blood vessels. Not having these large blood vessels at the macula improves the quality of central vision. Another feature of the central retina is the fovea, which appears as a dark spot around 2mm in diameter. This part of the retina is much thinner. The fovea marks the convenient anatomical reference point for the retina. This reference point has allowed to recognition of other parts of the retina. Nasal is part of the retina which lies closer to the nose, temporal near the temple, superior area of the retina above the fovea and finally inferior area of retina below the fovea.
Looking at the cross-section of the eye, the cornea does not get any supply from blood vessels but is surrounded by a fluid called the aqueous humor. The aqueous humor provides the cornea with nourishment. The lens is suspended by zonule fibres which are attached to the cilliary muscles which are further attached to the sclera forming a ring inside the eye. Lens changes shape allowing the eye to adjust its focus to see different objects at different distances. The lens divides the interior of the eye into two fluids firstly the aqueous humor and secondly the intreous humor which lies between the lens and the retina keeping the eyeball spherical [Bear et al., 2007]
Light passes into a medium where its speed is slowed and bends toward a line that is perpendicular to the border [Bear et al., 2007]. This is also seen when light hits the cornea from the atmosphere into the aqueous humor. The curve structure of the cornea allows the light to bend and passes through suffiently reaching the back of the eye in the centre of the retina. The cornea has a refractive power of 42 diopters meaning light will be focused for 2.4nm which is almost the same distance from the cornea to the retina. This power of refraction can be displaced therefore we would be seeing blurry vision for example when under water the water-cornea interface has limited focusing power and we see blurry when we open our eyes under water. However a scuba mask replaces the air-cornea interface therefore restoring refractive power of the eye.
The lens itself undergoes a process known as accommodation were the lens changes shape providing additional focusing power. Providing a further few dioptres to form a sharper image. As an object approaches the light rays are not parallel and diverge therefore greater refractive power is needed to get focus on the retina. To allow focus on the retina the lens accommodates itself. The ciliary muscle around the lens contracts and dilates during accommodation. Decreasing the size and tension of the muscle inside and the suspensory ligaments. This is all achieved by the lens elasticity. Due to this elasticity of the lens it becomes rounder increasing the curvature of the lens surface. Thus increasing its refractive power. When the ciliary muscle begins to relax it increases the tension of the suspensory ligament stretching the lens into a flatter shape [Bear et al., 2007]. Accommodation ability of the lens is altered during aging. As age increases focusing ability decreases. The pupil also contributes to the optical functioning of the eye. As it continuously adjusts itself to accommodate different levels of light throughout the day. This is known as papillary light reflex which is achieved by the involvement of the retina and neurons from the brain stem which control the muscles of the pupil. Papillary reflex has a feature known as consensual; when a beam of light is shined into one eye it causes constriction of pupils in both the left and right eye.
The innermost layer of the eye is the retina. The outer layer of the retina; the pigmented epithelium attaches to the choroids. The pigmented epithelium has a layer of cuboid cells with large amounts of pigmented granules in their cytoplasm [Brodal., 2010]. Following the pigmented epithelium are three more internal; photoreceptors and two layers with neurons. Photoreceptors are connected to bipolar cells which transmit signals to the retinal ganglion cells. Ganglion cells axons are connected to the optic nerve and signals travels into the nuclei of the diencephalon and the mesencephalon. On the other side of the pigmented epithelium opposite to the posterior to the ciliary body the pigmented epithelium extends to the edge of the pupil [Brodal., 2010]. The retina also contains amocrine cells and horizontal calls. Lateral inhibition occurs due to the presence of the horizontal cells as well as other functions. Photoreceptors of the retina are the only light-sensitive cells, other cells are either directly or indirectly influenced by light from the synaptic interactions with the photoreceptors [Bear et al., 2007]. Ganglion cells projects axons through the optic nerve therefore outputting information from the retina. No other cells within the retina have this ability. The axons of the ganglion carry visual information to the thalamus. Photoreceptor cells transmits synaptic inputs with the lateral interneuron's, the horizontal cell which then goes onto make synaptic connections with either the synaptic terminals of the photoreceptors or the bipolar neurons depending upon which species. Amocrine cells makes synaptic connection with the bipolar cells from which these cells receive inputs and also makes synaptic connections with the dendrites of ganglion cells. Feedback synapses between the cells of the retina are very common.
Photoreceptors are of two classes rods and cones which primarily named by the shape of the photoreceptors outer segment. At the outer segment of the light-sensitive portion of the cell light is absorbed and a process known as photo-transduction occurs. Stacks of disks are embedded in the cell membrane of the outer segment. Within these disks photoreceptor's light-sensitive pigments are located. Rhodospin is the photo-pigment which is contained within these disks in the outer segment. The outer segment of robs are long cylinder shape with many disks. As for cones the outer segment is short and tapered towards the distal end [Mathews., 2001]. Rods carry out scoptopic vision allowing vision in dim light. As the rods contain many more photo-pigments than cones they have a greater sensitivity for light about 1000 times more than cones. There are 120 million rods within the human eye. On the other hand there are only approximately 6 million cones in the human eye. Cones carry out photopic vision allowing us to see in bright light. This particular type of vision is sensitive to colours and gives very good clarity of the image we see. Cones have either one of the three photo-pigments within their folded membrane unlike the rods which contains them in there disks. A thin ciliary neck containing vestigial ciliary appraratus connects the outer segment to the rest of the photoreceptor cell. The non-light sensitive part of the photoreceptor is the inner segment. The inner segment contains the nucleus and other cellular organelles within the neurons. Synaptic terminal is formed by the other parts of the inner segment with the second-order horizontal and bipolar neurons. The total length of the photoreceptor is less than 100 µl therefore allowing the light from the outer segment to easily move through the photoreceptor to the synaptic terminal without needing to produce an action potential.
As photoreceptors do not produce action potentials light causes a graded change in membrane potential and rate of transmitter release onto postsynaptic neurons. This process of light travelling through the photoreceptor by generating an electrical response is known as phototransduction. Photo-transduction process corresponds to transduction of chemical signals into electrical signals. During a G-protein coupled receptor binding of neurotransmitter to the receptor activates the G protein altering the intracellular concentration of the cytoplasmic second messenger molecule. This alteration changes the conductance of the membrane ion channels either directly or indirectly which in turns alters the membrane potential. Within rod photoreceptors when light is emitted onto the eye, the light stimulates the photo-pigments within the disks of the outer segment in turn activating the G-protein coupled receptor leading onto activate an enzyme. Activation of the effector enzyme changes the cytoplasmic concentration of the second messenger molecule [Bear et al., 2007] leading onto the closer of the membrane ion channels thus changing the membrane potential. The resting potential of the neurons is about 65 mV which is very similar to equilibrium potential of K+. When we are in the dark the membrane potential within the outer segment of the rods is -30 mV. Dark current is seen within the neurons during darkness when positive charged ions move across the membrane. Positive charged ion being Na+ causes depolarization of the neurons when there is a steady influx thought-out the outer segment via sodium channels. The sodium channels are stimulated to keep open by its intracellular second messenger, cyclic guanosine menophosphate (cGMP). cGMP is readily produced by guanylyl cylase within the photoreceptor keeping the sodium channels open. When light is present it reduces the production of cGMP thus closing the Na+ channels and making the membrane potential more negative. Hence photoreceptors hyperpolarise in the presence of light. Photo-pigments within the outer segment of the rod photoreceptor absorbs electromagnetic radiation leading to hyperpolarizing of the neuron. Rhodopsin within the photoreceptors can be seen as a bound agonist. Opsin is a seven membered trans-membrane alpha helice which is very similar to the structure of the G-protein coupled receptor. Opsin is a typical receptor protein which has the bound agonist retinal. Retinal is the derivative of vitamin A. Absorption of light causes a process called bleaching, were the light alters the structure of retinal activating opsin. This changes the wavelengths absorbed by rhodopsin. Bleaching leads onto rhodopsin stimulating transducin a G-protien coupled receptor located in the disk membrane. Activation of the transducin in turn activates phosphodiesterase (PDE) an effector enzyme. The PDE breaks down the cGMP in the cytoplasm causing the Na+ channels to close causing hyperpolarization of the membrane. One function of this cascade is signal amplification. As numerous amount of G-protein coupled receptors are activated by a single pigment and a single PDE enzyme can break more than one cGMP molecule, this gives the ye and brain the ability to detect a single photon.
Looking at the photo-tranduction in cones, vision during day light depends solely on cones as when light is present rods are fully saturated due to the decreased amount of cGMP. During the day cones require more energy for the photo-pigments to become bleached. Process of phototransduction is very similar in cones and rods expect for the type of opsin present within the disks of the outer segment. Cones in the human eye contain one of three opsins, each having a different spectral sensitivity. These three opsins includes blue cone which is maximally activated by light at wave-length of around 430nm, green cones activated around 530nm and red cones activated around 560nm.
Perception of colour is dependent on the contribution of red, blue and green cones. Visual colour perception was first shown by Thomas Yand. He showed all colours as seen in the raindow plus including white could be produced by the correct ratio of the three different light-sensitive cones. This observation lead onto the theory we know as the young-helmholtz trichromacy theory. This theory suggests the brain assigns colour depending on the three different cone types. Also suggesting equal stimulation of the three cone types leads to the brain perceiving the colour white. One or more types of cone photo-pigment missing gives rise to different forms of colour blindness.
Short wavelength (SW) sensitive cone opsin gene also known as the S gene is located on chromosome 7. L and M genes are encoded on X-chromosome [Corroll et al., 2004] and are very similar both consisting of 6 exons which are separated by a long sequence of non-coding region called introns. Genes encoding L and M cones are 15.1 and 13.2 kilobases (kb) in length respectively. Between these two genes there is a 1.9kb difference in residue within intron number 1. The non-coding region separating the genes are 24kb long. Amino acid sequence encoding for S opsin consists of 348 animo acids which is similar to other vertebrate that encode opsin for violet and ultraviolet sensitive cone pigments.
L opsin and M opsin are 96% homologous therefore belong to the same phyiogenetic group of LW opsin [Bowmaker., 1998]. L opsin and M opsin both have an amino acid sequence of 364 residues, with only 15 different sites. This allows the approximate 30nm spectral difference of the two pigments favouring to substitution at these sites. Substitution in the membrane helices and of close proximity to the chromosome results in major effect to the spectral tuning or the pigment. Most significant effect on the chromosome are the replacement of charged for non-charged or polar for non-polar amino acids [Bowmaker., 1998]. There are three sites within the sequence which are likely to be involved implications these include helix 4 at site 180 and helix 6 at sites 277 and 285 which are responsible for 20-25 nm of the spectral difference. These sites are occupied by non-polar residues which include alanine at helix 4 site 180 and phenylalanine and alanine at helix 6 sites 277 and 285respectively. Within LW P562 they consist of three hydroxyl-bearing polar residues with a negative charge these include serine, tyrosine and threonine.
As L and M gene are so closely related in homology this region on X-chromosome is more susceptible to miss paring during meiosis resulting in crossing over between genes leading to deletion of the gene or replacement of the gene for another gene. Crossing over occurring within the genes itself results in a hybrid or chimeric gene which combines the region of L and M genes into a single gene. Formation of hybrid is what has been suspected to lead to anomalous colour vision in humans [Bowmaker.,1998 and Nathans.,1986]
Amino acids involved in spectral tuning their condons are located in exon 3 at site 180 and exon 5 for both 277 and 285 site. As site 277 and 285 are located on the same exon, the hybrid genes will greatly determine λ max of the visual pigment in repeats to whether exon 5 is from an L gene or an M gene. Exon 5 expressing L gene has a higher λ max compared to the M exon 5. Changes in exon 2 and 4 also show some degree of spectral shifts but only of a few nanometers. A spectral shift largely depends on which single amino acid substitution is occurring and for which opsin. Asenjo and collegues (1994) suggested that at site 180 if serine was substituted for alanine it caused a 2nm red shift in MW cone pigment and a larger shift of 7nm in LW cone pigment.
People with normal colour vision have some variation in colour matching. Analysis in Caucasian males should 40% of males require more red in their mixtures. Further analysis of these individuals showed that there is polymorphism existed in the L gene [Bowmaker, 1998]. Resulting form a single nucleotide difference in the gene causing a change in genetic code for amino acid on site 180. Within the population some people encoded for alanine at site 180 and other express serine. The correct arrangement of genes in the array has proven to be more difficult then expected. As the array is more complexed that just one L gene followed by one M gene.
Nathans et al (1986) found that L gene is always the first gene in the array followed by either one to four M genes however most individuals have two genes. Neitz and colleagues found that the array was much longer than expected and some individuals have almost 9 genes which may be hybrid. From this vast variation we do not know who may exhibit normal colour vision [Bowmaker.,1998]. Locus control region located 4kb upstream of first gene in the array helps the control of expression. Gene promotion depends on the folding or looping of chromosome which will bring the locus control region in contract with the single promoter region [Winderick et al., 1992]. Analysis of individual with blue cone monochromacy has shown the significants of the locus control region. Individuals with blue cone monochromacy have only functional SW cones and rods. Analysis of red/green gene array of these individuals showed the upstream length of the array was deleted including the locus control region in all cases. However in other individuals also suffering from blue cone monochromacy their condition was a result of gene deletions and point mutation in the genes leading to non-functional pigments.
Red/green colour blindness
As humans rely on three different light-sensitive cones within the retina long (L), middle (M) and short (S) wavelengths which donate red, green and blue light respectively. Humans and other old primates have a three-colour system therefore and known as trichromats. Functional loss in either of the cones results in dichromatic colour vision. A form of these types of abnormalities is red-green colour blindness.
Sex linked red/green colour deficiencies is correlated to the location of the L and M gene array on X-chromosome. These genes reside in a head to tail tandem array [Corroll et al., 2004]. There are two classes of red/green defects firstly, dichromat were complete colour blindness is seen in red/green and secondly, anomalous trichromats were there is reduced sensitivity to either red or green. A single visual pigment in present in the middle to long wave in dichromats. This pigment is either the LW or MW pigment deuteronomaly is the commonest form of anomalous colour vision. It affects 4% of men were they have reduced sensitivity to green light. Protonomaly is less common affecting only 1% of men. Protonomaly is when more red lights are required. Vast variation between the two forms of anomaly has been found. Some individuals suffer form a mild form or severe from. Also their precision of anomaloscope matches vary in respect that same individual make precise matches were as other accept a wider range.
Red-green colour blindness is quite common among the human population affecting 8% of males. These LW, MV and SW sensitive opsin genes were first cloned from humans by Nathans et al (1986). Thus allowing opportunity to explore the molecular genetics behind red/green colour deficiencies. Spectral tuning studies showed the spectral sensitivities of MS and LW photo-pigments in human are determined by A180, F277 and A285 [Yokoyama and Yokoyama., 1990; Netiz et al., 1991; Yokoyama and Radlwimmer., 2001]. One locus is responsible for the sensitivity difference between each cone. Both loci for red and green were mapped on the distal part of the X-chromosome on the q arm. And were seen to be linked closely to one another and also the glucose-6-phosphate dehydrogenase [Nathans et al., 1986]. Studies using models with loci responsible for inherited colour vision was shown to correspond to the genes that encoded for the apoprotein of the three cone pigments [Nathans et al., 1986]. This work was supported by study carried out by Ruston (1963, 1965) and Aplern and Wake (1997) who measured visual pigment absorption in a different number of living human eyes by the method of reflection densitometry their findings showed that dichromats lacked one of the three cones photo-pigments.
Nathans et al (1986) analysis for the red and green pigment genes using southern blots showed individuals with inherited red/green colour blindness had undergone rearrangemtn. These rearrangemt observed produced chimeric proteins which contained segments of red and green pigment [Asenjo et al., 1994]. As discussed earlier due to highly proximity of these x-linked pigments unequal homologues recombination of the two genes strongly influence the risk of chimeric pigments. Asenjo et al (1994) suggested there are 7 amino acids involved in the difference seen between the two pigments which include Ser (117) → Tyr, Ser (180) → Ala, lle (230) → Thr, Ala (233) → Ser, Tyr (277) → Phe, Thr (285) → Ala and Tyr (309) → Phe. These changes in the green pigment to amino acids found in the red pigment completely converts absorption spectrum of green pigment to the re pigment [Asenjo et al., 1994]. Numerous studies propose spectral difference between red and green colour vision is entirely due to residues at positions 180, 277 and 285. To further support this chan et al (1992) substituted hydroxyl-bearing amino acid side chains in rhodopsin in rods at these positions resulting in a red shift of the spectral absorption. Asenjo et al (1994) work suggests amino acids at position 116, 230, 233 and 309 are also contributed to a spectral shift.
Rhodopsin present in all three cones is encoded by a single gene on chromosome 3 which is expressed in all rod photoreceptors. Gene which expresses the blue cone opsin shows autosomal characteristics. As already discussed mapping of the genes showed genes which derived red and green opsin were adjacent and are approximately 98% identical. Due to the high similarity and very close proximity of red and green gene they are primarily influenced to frequent homologous recombination [Neitz and Neitz, 2000]. This alongside natural selection may have given rise to the wide range of variability in the red or green photo-pigment genes. The genes have undergone duplication of either red or green genes leading to many people having an extra pigment gene
Blue cone monochromatism
Blue cone monochromacy (BCM) is very rare x-linked disorder affecting less then 1 in 100,000 people. Affected males have normal functioning blue cones and rods but lack red and green cones function. Alternations in genes have been associated with BCM [Ayyagari et al., 2007]. As discussed earlier genes coding for these colour pigments are arranged in a tandem array on the X-chromosome. Thus readily undergo homologues recombination or gene conversion resulting in BCM. Deletions of control region upstream from red opsin gene can be related to BCM. These deletions have a 579-bp overlap region located 3-4 kb transcriptional start of red pigment gene [Reyniers et al., 1995]. Resulting in loss of expression of both red and green pigment genes [Nathans et al., 1986]. A common gene conversion is mutation of a thymine to cytosine at position 648 of green pigment gene [Winderickx et al., 1992]. The highly conserved disulfide bridge becomes disrupted when cysteine at position 203 is replaced by araginine (C203R) [Reyniers et al., 1995]. A247T in red pigment gene and P307L in red/green pigment hybrid gene point mutations have been associated with BCM [Nathans et al., 1993].
There are very rare incidents in which one or more of the cones are absent, this condition is known as achromatosia were colour vision is absent and people with this condition see the world as a black and white movie. Certain people may be affected by the inherited form called incomplete achromatopsia all so known as blue cone monochromacy. Incomplete achromatopsia is characterised by the loss of function of all the light sensitive cone photo-pigments.
Dichromacy affects about 1 in 4000 women [Netiz and Netiz., 2000]. In most cases of dichromacy loss of colour vision is due to the loss of genes encoding for one particular light sensitive cone photo-pigment. However some exceptions have been reported with rare cases of protonape were found to have an intact L photo-pigment in addition for M photo-pigment gene. This is due to the fact that genes for the L opsin interferes with the expression or function of the L pigment [Netiz and Netiz., 2000]. This sort of rare exception was also seen in a study of 100 school boys which were diagnosed colour vision defects. 12 boys were found to be deuteranopia but 2 out of the 12 had intact M photo-pigment genes [Netiz and Netiz., 2000]. Nucleotide suqencing showed that due to point mutation a cysteine changed to argentine at position 203 within the G-protein coupled receptor was responsible for the loss of function of M photo-pigment. This type of point mutation of the essential amino acid residue has been associated with incomplete achromatopsia and deuteanomaly [Nathans et al., 1989; Winderick et al., 1992 and Netiz and Netiz., 2000]. The cysteine residue within all G-protein coupled receptors is highly conserved and is an essential residue for forming disulfide bond in the photo-pigment molecule. These mutations were first seen in association with blue monochromacy [Nathans et al., 1986].
As cone photoreceptor diseases are vision limiting and many retinal diseases do not affect cones directly but eventually leading to cone loss due to these reasons gene therapy has become a major therapeutic approach. Kohl and colleagues (2002) reported families with achromatopsia showed protein-transcation mutations in the GNAT2 gene which is located on chromosome 1p 13. GNAT2 gene is responsible for the alpha subunit of transducin in cone phototransduction cascade which couples to the visual pigments. Alexander and Hauswirth (2008) study showed GNAT2 mouse model having one of the genetic form of human achromatopsia showed recovery of normal cone function and visual acuity with treatment of vector that supplied wild type GNAT2 protein to cones. Primary cause of achromatopsia has been linked to three mutations genes; CNGB3 a beta subunit of the cone cyclic nucleotide-gated cation channel, CNGA3 an alpha subunit of the cone cyclic nucleotide-gated cation channel and GNAT2. The Jackson laboratory discovered nautrally mutated mouse expressing mutations in CNGA3 and GNAT2 [Pang et al., 2010]. Recent studies carried out by Pang et al (2010) using adeno-associated virus (AAV) mediated gene therapy restored function within cones of the mouse models. Supporting the pervious work carried out by Alexander and Hauswirth (2008). Corrective gene therapy may be worthy for human suffering from achromtopsia and other related cone deficiency disorders.