Neurosensory Retina

Structure

• 9 layers (outermost to innermost). 10 if you include the RPE 
• Outer segments of the photoreceptors

• External limiting membrane: formed by Muller cells and separates the inner and outer segments of the photoreceptors from their nuclei/cell bodies

• Outer nuclear layer: photoreceptor cell bodies

• Outer plexiform layer: processes and synapses between photoreceptors, bipolar cells’ axons and horizontal cells.

• Watershed zone between the outer third of neural retina (mainly photoreceptors) supplied by the choroid and the inner two-thirds supplied by the central retinal artery.

• The most common site for fluid accumulation in cystoid macular oedema

• Inner nuclear layer: cell bodies of bipolar cells, horizontal cells, amacrine cells and Muller cells

• Inner plexiform layer: synapses and processes between bipolar cells, amacrine cells and ganglion cells

• Ganglion cell layer: cell bodies of ganglion cells
• Nerve fibre layer: axons of ganglion cells
• Inner limiting membrane: formed by foot processes of the Muller cells.

Macula

• Yellowish oval
• 3mm lateral and 0.8mm inferior to disc
• 4.5mm in diameter (3-3.5 disc diameters)

• Yellowish due to lutein and zeaxanthin (xanthophyll compounds) in the retinal layers (but not in the RPE)

• Lutein is dispersed throughout the retina

• Zeaxanthin is concentrated at the fovea and is found in higher concentrations in cones compared to rods

• Fovea centralis: 1.5mm diameter (same as the disc)
• Maximal cone density here: 150000/mm2 with a 1:1 ratio with ganglion cells

• Floor of fovea is foveola: contains only photoreceptors (bipolars and ganglion cells are displaced laterally)

• 300 micron diameter foveal avascular zone (laser should be avoided in this area)
• In the macula the RPE are different
• Taller
• Thinner
• Contain more and larger melanosomes

Photoreceptors

• Derived from clinical ependymal cells of neuroectoderm origin
• 120 million rods
• 150000/mm2 in perifovea (absent from fovea), decreasing to 30000/mm2 at periphery
• 6.5 million cones
• 150000/mm2 at fovea, 5000mm2 in 10 degrees out-with  fixation.
• Nasal and superior retina contains most cones
• Cone density is 10x lower than fovea at only 1mm away from it
• Red and green-sensitive cones predominate 
• There are no blue cones at the fovea
• Red and green cones have highest visual acuity in bright light

• Basic structure:

• Cell body with nucleus: separated from inner and outer segments by external limiting membrane (adherens junctions)

• Inner segments are equal or larger than outer segments so that light is channelled to the outer segment

• Extended synaptic terminal known as ‘inner fibre’ leads to spherule (rod) or pedicle (cone: which has more invaginations and neural connections than the rods’ spherules)

• Inner aspect of inner segment is called the Myoid: numerous Golgi and rough ER
• Predominantly involved in synthesis
• Outer aspect of inner segment is called the Ellipsoid: numerous mitochondria.
• Predominantly involved in energy production

• Modified cilium connects inner to outer segments: there are pairs of microtubules within this structure

• Outer segment comprise ‘discs’ containing visual pigment.
• Rhodopsin-containing discs in rods are surrounded by plasma membrane

• Photopsin containing discs in cones have no membrane (ie. are continuous with the interphotoreceptor space).

• Note: outer segments do not contain mitochondria
• Rod length: 100-120 microns
• Cone length: 60-75 microns 
• Outer segment discs are produced at the base near the cilium

• Cytoplasm filled plates are formed by lipid and protein accumulations inside an out-pouching of the outer segment membrane which contains opsins

• The plates expand to the width of the photoreceptor outer segment
• Rim proteins are added at the cilium

• Include peripherin, ROM1, ATP-binding cassette (ABC) transporters and the cystic fibrosis transmembrane regulator (CFTR)

• In rods, the upper and lower membranes close and the discs are internalised

• peripherin/rds molecules and rom-1 molecules work at the disk lamellae to provide stability

• There are about 1000 sacs/discs within a rod outer segment with 1 million rhodopsin molecules in each sac

• Cone pedicles: 12 indentations, each with 3 neuronal terminals (“triad”)
• Central terminal: midget bipolar cell axons
• Lateral terminals: horizontal cell processes
• 3 cone types (according to spectral sensitivity and the type of photopsins they contain):
• Blue (short wavelengths)
• Green (medium wavelengths)
• Red (long wavelengths)

• Shedding: the apices of the photoreceptor outer segments slough off
• Rods: shedding maximal 1 hour after light exposure
• Cones: shedding maximal 2-3 hours after
• Regulated by melatonin (see below) therefore circadian cycle

Interphotoreceptor matrix (IPM)

• Space between the photoreceptor outer segments and the RPE: from outer limiting membrane to the surface of the RPE cell

• Consists of proteins, glycoproteins, GAGs, and proteoglycans (chondroitin)
• Note: there is no physical connection between photoreceptor outer segments and RPE

• Function:
• Retinal attachment and adhesion
• Molecular movement
• Facilitation of phagocytosis

• Interphotoreceptor retinoid binding protein (IRBP) accounts for 70% of the soluble proteins in the IPM and is synthesised by photoreceptors

Rhodopsin

• Freely diffusible membrane protein similar to alpha- and beta- adrenergic receptors

• Red pigment: reflects red light and so appears red but is actually most sensitive to green light (510nm) and least sensitive to red.

• Synthesised in the ER and Golgi of the inner segments: regenerated when eyes are closed
• Constructed from:
• The protein opsin 
• 348 amino acid protein, seven-turn alpha helix
• A molecule of vitamin A (retinol) that lies between loops of the rhodopsin molecule
• Acts as the chromophore (molecule providing colour)
• Retinal is the light absorbing form of vitamin A
• Integrated into the outer segment plasma membrane

• The ligand for rhodopsin (11-cis-retinal) is bound in the dark and dissociates when activated by a photon of light (see below)

Bipolar cells

• Transmit signals between photoreceptors and ganglion cells (ie these are the first order neurons of visual pathway)

• Located in inner nuclear layer
• At the fovea the ratio of cones:bipolars:ganglion cells is approx 1:1:1
• At periphery, rods:bipolars is approx 50-100:1
• There are 9 types of bipolar cells
• Cone bipolars:
• Can be divided into on- and off-bipolars utilising glutamate

• Some cone bipolars only synapse with L cones and others only with M cones to provide colour vision

• 5 Diffuse cone bipolars.

• These combine information between numerous cones. Dendrites fan out to clusters of 5-7 cone pedicles with much overlap

• 3 Midget cone bipolars:

• Invaginating midget: smallest type. Synapses with one cone pedicle to form the central process of the “triad” (cone:bipolar:ganglion). 1:1 ratio centrally with cones

• Flat midget: connects one cone to a single midget ganglion cell. Similar to invaginating midgets but don’t penetrate pedicle as deeply

• Blue cone specific midget

Ganglion cells

• 1.2 million (100:1 ratio of photoreceptors to ganglion cells)
• Cell bodies in GCL, but some ‘displaced’ cells may be found in INL

• Axons synapse in lateral geniculate nucleus of the thalamus

• Axons have a sheath provided by glial cells
• Axons are myelinated by oligodendrocytes after they exit the lamina cribosa
• 8 layers of ganglion cells at the fovea, but are absent from foveola

• Histology:
• Large cell body
• Large Golgi
• Lots of Nissl substance (rough ER)
• Contain xanthophyll carotenoid in cytoplasm
• Midget ganglion cells: driven by L or M cones
• Synapse amacrine cells or single midget bipolars (exclusively)
• Concentrated at the fovea: provide high acuity here

• Aka P-cells: project to the parvocellular layer of the LGN

• Provide high spatial resolution and colour vision: what we see
• Diffuse (parasol) ganglion cells: driven by S cones
• Synapse with all bipolars except midgets
• Less concentrated at the fovea, more so in the periphery

• Aka M-cells: project to magnocellular layer of the LGN

• 5 subtypes
• Provide movement detection: where we see it
• Intrinsically photosensitive retinal ganglion cells (ipRGCs)
• Contain melanopsin: highest sensitivity in the blue-violet range
• 5 subtypes

• Contribute to circadian rhythm, sleep cycle regulation, pupillary light reflex and detection of brightness

Horizontal cells (association neurone)

• Antagonistic interneurones: have an inhibitory effect on photoreceptor signalling by using GABA

• Type A: large, axonless, only connects cones
• Type B: smaller, single axon connects rods but dendrites connect cones
• Nuclei in INL
• Processes in OPL (hence they ‘face’ outwards)

Amacrine cells (association neurone)

• Dendritic trees synapse with bipolars and ganglion cells in the IPL
• Cell bodies in INL (hence they ‘face’ inwards)
• Large oval shaped cells
• 25 different types
• Release GABA, dopamine and ACh (mostly inhibitory effect on signalling)
• Amacrine II cells: release glycine (inhibitory) and link rods to ganglion cells
• Starburst amacrine cells: release ACh
• Dopaminergic amacrine cells are post-synaptic to bipolar cells

Astrocytes (glial cell)

• Scaffold around vessels and neurones
• Lie perpendicular to Muller cells and neurones
• Gap junctions between cells
• Prevent unwanted signals/effects on neighbouring cells

Muller cells (glial cells)

• Main supporting cell in the retina: similar to oligodendrocytes in CNS
• Nuclei in the INL
• Extend between ILM and ELM
• ILM formed by basement membrane and processes

• ELM formed by junctions between Muller processes and photoreceptors. Encloses the subretinal space

• Provides a sheath for vessels, cell bodies and processes 
• Numerous ER and microtubules (so contribute protein production)
• Contains proteins that bind retinaldehyde
• Produce insulin and growth hormone
• Buffer ions in the extracellular space

Microglial cells

• Mononuclear phagocytes
• Antigen-presenting cells
• Activated in response to retinal injury

Retinal metabolism

• Highest rate of aerobic glucose consumption of any tissue
• Respiratory rate of double that of brain tissue

• Half of the retinal respiratory rate is due to the ellipsoid region of the photoreceptors (which have numerous mitochondria)

• Lactate accumulates in the retina regardless of oxygen availability

Retinal glucose metabolism

• >80% of glucose use is by photoreceptors

• Insulin-independent tissue: glucose transport into cells depends on its concentration and not on insulin

• However endothelial cells in the retina have numerous receptors for IGF-1 and IGF-2 

• GLUT1 and GLUT3 transporter proteins allow diffusion
• Muller cells: store energy and contain high glycogen levels

• Glycolysis:

• Despite high aerobic glucose consumption, there is a large proportion of lactate conversion via glycolysis (unlike in other tissues)

• Glycolysis occurs in cytoplasm and produces pyruvate and lactate from glucose: providing 2 ATPs

• Phosphofructokinase and hexokinase catalyse the rate-limiting steps in glycolysis
• Kreb’s citric acid cycle
• Metabolises pyruvate providing 36 ATPs
• Requires oxygen

• The six carbon sugar citric acid is converted to a four carbon sugar yielding the hydrogen ions and ATP (3 ATPs for every 2 released hydrogen ions)

• Occurs on the inner mitochondrial membrane
• Pentose-phosphate pathway
• Alternative route of glucose metabolism
• Produces ribose, which is needed for RNA and DNA nucleotides, and NADPH
• Reversibly linked to the glycolytic pathway
• Gluconeogenesis
• Production of glucose from non-carbohydrate precursors
• Influenced by glucagon
• Protein utilisation: amino acids are transaminated 
• Fats: glycerol is converted to glucose by dihydroxyacetone phosphate
• Takes place mostly in the liver

Retinal neurotransmitters

• “Putative” (ie unique) neurotransmitters 
• Synthesised and stored in the presynaptic cell
• Released when the presynaptic cell is stimulated

• Produce hyperpolarisation/depolarisation in the post-synaptic neurone rather than an action potential (except in ganglion cells)

• Removed from the synaptic cleft by degradation or by reuptake into the presynaptic cell

• Feedback pathways in the retina tend to involve inhibitory neurotransmitters
• Glutamate:
• Major excitatory neurotransmitter in the retina
• But also has some inhibitory action on cone photoreceptor-ON bipolar synapse
• Receptors for glutamate on all secondary and tertiary retinal neurones

• Receptors may be ionotropic (work on Na/K, or Ca permeability) or metabotropic (G-coupled receptors)

• Recycled from synaptic cleft: diffuses back into the presynaptic cell (but is not degraded)

• ON-bipolar:

• Glutamate released from presynaptic cell in the dark and binds to metabotropic receptor on the ON-bipolar producing hyperpolarisation. Bipolar Na channel stays closed

• Light exposure to the photoreceptor leads to cessation of glutamate release which causes cGMP to build in the bipolar cell. Na channels open, depolarising and allowing a signal to transmit.

• OFF-bipolar:

• Glutamate binding to the ionotropic receptors on the OFF-bipolar produces hyperpolarisation in light conditions (the reverse of the above)

• GABA:
• High concentration throughout retina
• Main inhibitory neurotransmitter

• Synthesised from glutamic acid (ie. derived from glutamate) decarboxylase, an enzyme present in amacrine cells

• Found in horizontal, amacrine and Muller cells

• Acetylcholine: used by amacrine cells for motion detection (major excitatory neurotransmitter) and input to bipolar cells

• Dopamine: also found in amacrine cells and has calcium dependent release in response to light. Acts on horizontal cells and contributes to light-dark adaptation.

• Catecholamines act on the RPE

Melatonin

• Neuropeptide hormone released by photoreceptors and acting locally on the retina
• Released in a circadian fashion

• The suprachiasmatic nucleus receives input from the retina via the retinohypothalamic tract

• The circadian rhythm cycle is controlled by pacemaker cells in the nucleus which influence melatonin production

• N-acetyl-transferase converts serotonin to melatonin

• This enzyme is denatured by light, therefore serotonin increases in light conditions and melatonin in dark conditions

• Inhibited by dopamine D2 receptor (melatonin and dopamine work antagonistically)
• Dopaminergic amacrine cells regulate melatonin synthesis in response to light
• Contributes to photoreceptor disk shedding (in a diurnal manner)
• Has antioxidant activity as a hydrogen peroxide scavenger