## 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

In the macular region, this layer is known as the fiber layer of Henle where it becomes thicker and more obliquely orientated to become horizontal and parallel to the retinal surface at the fovea. This accounts for the radial/star-shaped pattern of exudate seen here eg. in neuroretinitis (cat-scratch).

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)

Histologically defined as the retinal area where the ganglion cell layer is more than one cell layer thick: two-three sub-layers

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)

There are no rods at the fovea

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

Gass system of macular hole formation (expanded in Part 2 package)

83% idiopathic, 15% traumatic
4 stages of formation described by Gass based on theory of centripetal tangential traction by the vitreous at the fovea

1a: tractional elevation of the foveola with visible yellow dot
1b: enlargement of the tractional detachment with yellow ring
2: full thickness retinal defect <400micrometres
3: full thickness retinal defect >400micrometres
4: stage 3 with additional PVD

## 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

Rod outer segments are completely recycled over 10 days: they travel towards the tip and are phagocytosed by the RPE in a circadian cycle
Cone discs have a longer lifespan and are not phagocytosed in a circadian way.

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)

Abundant vitamin A is required for normal vision and must be supplied exogenously.

## 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

Contribute the b-wave of the ERG

## 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

The macula is defined as the area with more than one layer of ganglion cells

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

Proliferation of astrocytes contributes to PVR

## 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