Student I.D. Number: 8931194

B.Sc. Neuroscience

Supervisor Name: Andreas Prokop

COMPARE INSECT AND MAMMALIAN VISUAL ORGANS

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Introduction

Eyes are a marvel of evolution. They are perhaps some of the most complicated and intricate

structures in nature, the advancement of which has been a hugely powerful driving force within the

arms race of predator-prey relationships throughout natural history. The modern world as we know it

has been shaped to exploit our ability as humans to see well, and the role of vision as our primary

means of sensual perception. The ability to detect colour, motion and contrast is one we generally

take for granted, but the very means and mechanics of doing so utilise one of the most complex

neural systems in the body, and can show radical discontinuity between species.

Drosophila melanogaster is an almost universal model system within the study of biology. The relative

simplicity of its nervous system, coupled with the vast wealth of knowledge available on its genetics,

have proven to be of vital importance in unravelling many biological mysteries, including vision. The

beauty of Drosophila mutants and genetics is the opportunity they afford us to examine different

facets of vision separately, and so enable the assimilation of information in a bottom up, fairly

reductionist fashion (van Swinderen et al., 2013). One great revelation that has come from such

research is the common basis for eye development among multiple species, including Drosophila and

mammals. The Pax6 gene has been shown to induce ectopic eye formation in a range of insects and

vertebrates (Gehring, 2005). In humans, this gene encodes the paired box 6 (Jordan et al., 1992)

protein which is an embryonic transcription factor. Highly conserved among bilaterian species, it has

been shown to code for identical proteins in mice and humans, and even induce eye development in

Drosophila (Gehring and Ikeo, 1999). This common basis for development is further highlighted by

functional homology in the gene products of Drosophila Pax6 orthologues and vertebrate Pax6

isoforms. Examples include the eyeless gene (named for its phenotype), and the canonical vertebrate

Pax6 isoform, or between the eyegone gene, and the vertebrate 5a isoform of Pax6.

The course of natural history has produced myriad forms of visual apparatus, which have taken a

range of familiar and alien forms over the evolutionary time. Very early photoreception in planaria

utilised primitive eyecups, in which heavily pigmented cells screened photoreceptive cells from light in

all but one direction. These ‘proto-eyes’ would have had little use in discerning the direction of light,

merely its presence or absence. Slightly more advanced mirror eyes function similarly to a reflective

telescope, reflecting and focusing incoming light onto an array of photoreceptors, each of which acting

as an individual pixel. At the more advanced end of the spectrum we find such lens and compound

eyes as are common to many of today’s mammals and insects and that will be the focus of this

review.

The compound eye of Drosophila is perhaps the best understood visual organ after our own eyes,

and has played a massive role in taking our understanding of vision to the molecular and genetic

level, furthering earlier work carried out in larger flies. The primary aim of this review is to illustrate

and describe some of the fundamentals of fly and mammalian visual anatomy and optic pathways.

Comparing Phototransduction in vertebrates and flies:

Visible light travels as an electromagnetic wave and travels as an amalgamation of wavelengths,

frequencies and energies. The highest energy visible is blue shifted, becoming visible at a wavelength

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of approximately 380-400 nm, through to low energy red light that ceases to become visible at

approximately 750-760 nm. Phototransduction refers to the ability of photoreceptor cells to convert

this electromagnetic energy into an electrical signal which can be transmitted by the nervous system.

Due to the exquisite nature of vision, and the organs that permit it, slightly different mechanisms of

transducing energy from light to electrical have evolved in the eyes of the mammals and insects.

In either the human or fly eye, there is a fundamental principle, central to photoreception. This is the

pairing of the endogenous opsin receptor protein and its ligand; the chromophore retinal. Whether in

rods, cones, or invertebrate photoreceptor cells, these pigments exist covalently pre-bound to the 11-

cis isomer of retinal (Koutalos and Ebry, 1986). Photobleaching of this chromophore, by incoming

photons, converts it from antagonist form to the potent opsin receptor agonist: all-trans-retinal. This

photoisomerisation of retinal is the only means of light input into the phototransduction cascade, but

the resulting conformational change activates a downstream heterodimeric G-protein signalling

cascade (Arshavsky et al., 2002). All type II (or animal) opsins are themselves the receptors that

become activated by light, and constituents of a large superfamily of G-Protein coupled receptors

(GPCRs) (Feuda et al., 2012).

Different photoreceptors though, use different endogenous pigments. Each of the ommatidia in a

Drosophila’s compound eye stochastically expresses three of a possible five opsins (Gao, 2008). In

the fovea, humans express three cone opsins, or photopsins, known as short (S), medium (M), or

long (L) wave sensitive, respective to the wavelengths to which they are maximally absorptive. The

peak sensitivities of cone opsins are approximately 430 nm for S cones, 530 nm for M and 560 nm for

L (Solomon and Lennie, 2007). In vertebrates, the opsins are localised in the outer segments of

photoreceptor cells, reaching concentrations of up to 500 µM in these compartments (Pugh and

Lamb, 2000). They are found in stacks of densely packed membranous discs in rods, or in multiple

invaginations of the plasma membrane in cones (Burns and Arshavsky, 2005),

Rhodopsin is the photoreceptive pigment of the non-colour sensing rod cells that populate the

periphery of the human retina, and was the first to be both characterised (Kuhne, 1879) and

crystallised (Placzewski, 2000). The conformational state of the photobleached rhodopsin is also

referred to as metarhodopsin II, and is induced within merely a millisecond (Burns and Arshavsky,

2005 and Arshavsky et al., 2002) of photon incidence. In this state, it gains the capacity to activate the

photoreceptor specific G-Protein transducin, by catalysing a GDP for GTP exchange on the alpha

subunit of transducin (Fung et al, 1981). This triggers the rapid dissociation of the transducin trimer

(alpha, beta and gamma subunits) from the metarhodopsin complex, and subsequently of the now

GTP bound alpha subunit from the beta-gamma dimer. The now free alpha subunit exerts a

disinhibiting action on the effector enzyme, phosphodiesterase (PDE), through interaction with the

enzyme’s regulatory gamma subunit (Leskov et al., 2000). Activation of PDE quickly decreases the

intracellular concentration of cyclic guanosine monophosphate (cGMP), through its hydrolysis, to form

5’GMP. Subsequent cessation of plasma membrane bound, cyclic nucleotide gated cation channel

activation causes hyperpolarisation of the photoreceptor cell and termination of the so called ‘dark

current’. Thus, presynaptic glutamate release from the photoreceptor cell is decreased in response to

light. Signal amplification is achieved at multiple levels of this pathway, initially by the activation of

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multiple units of transducin by a single molecule of photoisomerised pigment, but additionally by the

massively high catalytic activity of PDE (Arshavsky et al., 2002). The resultant rate of transducin/ PDE

activation by individual rhodopsin molecules can reach 120-150 catalytic subunits per second (Leskov

et al., 2000), culminating in a system sensitive enough to detect single photons (Hecht et al., 1942).

Intracellular cGMP level restoration takes place in a calcium dependent manner and is undertaken by

guanylate cyclase and a number of guanylate cyclase activating proteins (GCAPs) (Placzewski et al,

2004)

Inactivation of the photoresponse is achieved via triple phosphorylation of metarhodopsin (Kennedy et

al., 2001) by rhodopsin kinase, in a calcium and recoverin regulated manner (Chen et al., 1995),

followed by arrestin capping (Vishnivetskiy et al., 2000). Inactivation of PDE follows GTP hydrolysis

by the transducin alpha subunit and its subsequent re-association with the beta and gamma subunits,

in a process accelerated in rods by the RGS9 protein (He et al., 1998). This mechanism of transducin

inactivation is approximately 1000 times faster than would be expected by natural thermal decal of

metarhodopsin alone (Hofmann, 2000). Cone cells achieve more rapid rates of dark adaptation and

pigment regeneration than do rods (Lamb and Pugh, 2004). This is thanks to faster thermal decay of

excited photopigments (DeGrip and Rothschild, 2000) and an additional enzyme based opsin pigment

regeneration pathway, surplus to the normal visual cycle present in rods, (Mata et al., 2002).

The shared aim of maximising the availability of phototransduction machinery results in a common

pattern of compartmentalisation in mammals and insects. However, unlike mammals, this is achieved

using tightly packed microvilli that collectively form a rhabdomeres of approximately 100 µm in length

(Hardie and Raghu, 2001). The different vertebrate and invertebrate opsins also share topology, but

differ by a single amino acid: the glutamate 113 position in vertebrate opsins is replaced by a tyrosine

in invertebrate visible light sensitive pigments, or by phenylalanine in UV pigments (Yarfitz and

Hurley, 1994). Furthermore, photopigment isomerisation in mammals catalyses cyclic nucleotide

exchange on the transducin G-Protein. In insects this GDP for GTP exchange occurs on the Gq G-

Protein isoform. Upon subsequent dissociation, the active, GPT bound alpha subunit stimulates the

effector enzyme phospholipase C (PLC) (Hardie and Raghu, 2001). This enzyme mediates the

breakdown of membranous phosphatidylinositol- 4, 5- biphosphate (PIP2) into diacyl glycerol (DAG)

and inositol triphosphate (IP3). The precise mechanism by which intracellular signalling downstream

of this point leads to the opening of membranous, cation permeable channels and subsequent

depolarisation of the photoreceptor cell remain unclear. This mechanism is also likely to differ

between different invertebrate species, since intracellular IP3 dependent calcium release is

considered essential to photoreception in Limulus (Nasi et al., 2000), but IP3 receptor mutations in

Drosophila leave phototransduction unaffected (Raghu et al., 2000).

Thus, the mechanism that leads to the opening of light activated Transient Receptor Potential (TRP)

and Transient Receptor Potential-like (TRPL) channels in Drosophila, which mediate photoreceptor

depolarisation (Hardie, 2001), may involve any combination of a plethora of secondary messengers.

These may include but not be limited to IP3, PIP2 or DAG. Via DAG lipase, DAG is a precursor for the

synthesis of polyunsaturated fatty acid (PUFAs), which have been shown to activate both TRP and

TRPL channels (Chyb et al., 1999). However a direct link here is disputed by virtue of PUFA

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instigated activation of light sensitive channels via ATP depletion (Agam et al., 2000). Moreover

though, mutations in the gene encoding DAG kinase (rdgA mutants) lead to drastic retinal

degeneration and an unresponsiveness of photoreceptors to light (Hardie, 2001) through

constitutively active TRP channels. Functionally, the DAG kinase enzyme depletes DAG

concentrations intracellularly and aids in the re-synthesis of PIP2, and the effects of the rdgA mutation

may implicate it in response termination (Hardie, 2001). More recently, Agam et al. (2004) report that

it may be the collective actions of Ca2+

ions and protein dephosphorylation that cause the activation of

TRP and TRPL channels when the photoreceptor cells of Drosophila depolarise in response to light.

Response termination in the fly, as in mammals, requires the regeneration of the non-photoexcited

form of the visual pigment. However, the form of metarhodopsin present in the dipterian compound

eye is thermostable, unlike in vertebrates, meaning that Drosophila metarhodopsin can be constantly

reconverted back to rhodopsin by exposure to ambient, longer wavelength/ red shifted light (Hardie

and Raghu, 2001). The transparency of Drosophila’s retinal screening pigments to long wavelength

light for this purpose is the origin of the fly’s natural red eye colour (Hardie and Raghu, 2001). This

time-saving strategy greatly increases the temporal resolution of the fly’s eye when compared to our

own, able to follow a flicker frequency of up to 300 Hz (Laughlin et al., 1987). Conversely, the signal

amplification - response speed trade off in human eyes limits our temporal resolution to as low as 10

Hz (Hardie and Raghu, 2001), meaning any light stimulus flickering at a higher frequency than this will

be perceived as constant.

Comparative anatomy of mammalian and insect eyes:

The eyes of mammals and insects differ from the top down, starting with their appearance. The

human eye has a very distinct and characteristic pupil, created by surrounding pigmented cells, where

light is collected and focused, primarily onto the fovea. Drosophila lack a pupil, or indeed any form of

master light collecting aperture. Instead, and as can be seen with the naked eye, the surface of the

compound eye is covered in perfectly tessellated hexagonal ommatidia. Each ommatidium is an

individual lens system and gathers light information from a distinct region of visual space.

The non-uniform distribution of photoreceptors in the human retina creates zonal functionality

between the fovea, parafovea and the periphery of the retina. Colour vision and generation of a sharp

image is undertaken at the fovea, while non-colour vision, changes in low level light intensity and

peripheral motion detection are undertaken by the cone based regions of the retina. Whilst five

subclasses of vertebrate opsin exist, one rod and four cone (Okano et al., 1992), evolution has seen

the loss of all but two in mammals, which retain only one long and one short wavelength sensitive

opsin (Solomon and Lennie, 2007). However, the duplication of a green cone opsin gene on the X

chromosome of old world primates has seen new wold primates re-evolve a third opsin gene. This

has occurred due to the accumulation of mutations on the duplicated gene shifting the spectral

sensitivity of its product (Jacobs and Rowe, 2004).

The ability of the eye to focus light comes from its refractive power. Most of this refraction takes place

at the air to cornea interface, with the cornea itself having a refractive power of approximately 42

dioptres, but the lens can add up to another 12 dioptres (Bear et al., 2007).

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The human retina has a laminar structure, with the photoreceptors synapsing onto bipolar cells in the

outer plexiform layer (OPL), which in turn synapse onto the ganglion cells in the inner plexiform layer

(IPL). Cells of the ganglion cell layer eventually converge to form the optic nerve, which creates a

blind spot at the rear of the retina where it exits. Amacrine cells also synapse in the IPL, with

ganglion, bipolar and other amacrine cells. The functions of the many subtypes of amacrine cell

include, but are not limited to include lateral inhibition, detection of directional motion, modulation of

light adaptation (Balasubramanian and Gan, 2014) and control of sensitivity adjustment in scotopic

vision (Marc et al., 2014). Horizontal cells also provide lateral connections within the retina, synapsing

in the OPL. These GABAergic interneurons prevent signal transfer between cone and bipolar cells

when hyperpolarised themselves.

Cells in the retina have their own receptive fields; areas of visual space in which a stimulus will elicit a

change in the firing of that cell. For photoreceptors, these are cone shaped volumes of visual space,

but those of bipolar and ganglion cells extend beyond the photoreceptors immediately above them in

the retina, thanks to horizontal and amacrine cells, which create the concentric centre-surround

receptive fields of bipolar and ganglion cells (Kuffler, 1953). On and off centre bipolar cells depolarise

in the presence of either dark or light stimuli respectively, at the centre of their receptive fields when

compared to the periphery (Paradisio et al., 2007). This creates two, parallel pathways of visual

information transfer within the retina (on and off pathways), but these do not necessarily show

symmetrical characteristics (Pandarinath et al., 2010). The on pathway utilises sign inverting

synapses between the cones of the receptive field centre and their contacting bipolar cell, while the

same synapses in the off pathway use sign conserving synapses. Hubel and Wiesel (1962) first

characterised the way in which these simple receptive fields of the retina are integrated to form

complex receptive fields in higher areas of the visual system.

The nature of the insect retina is far more uniform than that of humans and other mammals. The

compound eye of a Drosophila is comprised of between 700 and 800 regular facets, each of which is

the outermost face of a long thin cone of photoreceptor, pigmented and support cells that form the

ommatidium. The individual ommatidia each have features that would only be found singularly in a

mammalian eye, such as a cornea or lens system. Within an ommatidium, two distinct types of

photoreceptor are expressed; the inner and outer photoreceptors, so named for their respective

positions within the main column of the ommatidium. The six outer photoreceptors are termed

receptors R1-6, and are peripheral to the inner R7 and R8 photoreceptors, all of which are encircled

by a capsule of pigmented cells that give incoming photos directionality. The opsins of the insect eye

are localised to folds of the cell membrane called the rhabdomere (Hardie and Raghu, 2001).

As in the mammalian retina, a number of opsins are expressed in Drosophila, but these are distinct

from their mammalian visual counterparts, and instead better resemble the non-visual light sensitive

vertebrate pigment; melanopsin (Provencio et al., 1998). Photoreceptors R1-6 constitutively express

the Rh1 opsin (O’Tousa et al., 1985), which gives them a broad spectral sensitivity (Hardie, 1979).

The inner photoreceptors R7 and 8 are stacked on top of one another in the centre of the

ommatidium, with R7 being positioned ‘on top’ or closer to the exterior of the eye. R7 expresses one

of the two opsins Rh3 and Rh4, while R8 always expresses either opsin Rh5 or Rh6. The Rh3/4

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opsins are UV sensitive, while the Rh 5 and Rh6 have blue and green spectral sensitivites,

respectively (Salcedo et al., 1999). The outer photoreceptors of the Drosophila eye are widely

considered to be achromatic (Gao et al., 2008) and mediators of movement detection (Yamaguchi et

al., 2008). On the other hand, photoreceptors R7 and 8 are presumed to form chromatic channels that

contribute to the generation of spectral preference behaviours (Heisenberg and Buchner, 1977).

While the human retina undertakes a huge amount of initial processing of visual information, that

same computing in the fly begins to take place in the four layers of optic neuropil that make up the

higher centres of the insect’s visual system. Here, the uniform columnar organisation of the

compound eye is maintained through multiple layers of visual processing in Drosophila (Meinertzagen

and O’Neil, 1991), while downstream representation of the mammalian retina is warped in favour of

the fovea, creating anisotropy in the visual cortex (Blasdel and Campbell, 2001).

Comparing visual processing and circuitry:

Given that the brains and visual processing centres of some mammals are larger than the entirety of a

Drosophila, the degree of visual processing that can be undertaken in mammals greatly exceeds the

capacity of the fly to do the same. However, in both cases, once visual information physically leaves

the eye, it must undergo computation before it can be used to shape behaviours.

Visual information is passed out of the human eye by the axons of retinal ganglion cells (RGCs),

which cluster bilaterally to form the optic nerves. Information then decussates at the optic chiasm, at

which point; fibres form the temporal regions of the retinae project to the ipsilateral hemisphere of the

brain, while fibres originating in the nasal regions of the retinae proceed to the contralateral

hemisphere (Kandel et al., 2012). The first level of higher processing is the lateral geniculate nucleus

of the thalamus (LGN), part of the geniculostriate pathway. In humans and other primates, the LGN

consists of six layers of cell bodies, referred to as layers 1-6 ventrally to dorsally. The ventral layers (1

and 2) are comprised of magnocellular (M) cells, which receive input from M type RGCs, whereas the

dorsal layers three through six are parvocellular (P) and are fed by P type RGCs. On the ventral side

of each of these six principle layers exists an additional koniocellular layer. These koniocellular (K)

neurons are on average the smallest of the visual relay cells and express calbindin as their calcium

binding protein, as opposed to the M and P cells which express parvalbumin (Xu et al., 2001). The

physiological properties of the magno- and parvocellular systems have led to hypotheses that they

may comprise distinct extrastriate visual streams. For example, the parvocellular preference for higher

spatial frequency stimuli, and greater sensitivity to chromatic contrast have strongly implicated these

cells in the processing of detail and colour (Livingstone and Hubel, 1988). Similarly, M cells express a

preference for higher temporal frequency stimuli and are thought to form an achromatic pathway. K

cells also have distinct properties and characteristics. For instance, these cells receive input from a

further, different subclass of RGC (the nonM-nonP cells). This pathway was also been implicated in

chromatic processing when Martin et al. (1997) demonstrated that K cells received direct input from

blue/yellow contrast specific, on pathway ganglion cells. This finding also corroborated Livingstone

and Hubel’s 1988 proposition of a role for cytochrome oxidase blobs in chromatic processing, since

these are where the K cell axons project.

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The aforementioned cytochrome oxidase blobs are one of a number of features of the visual cortex

that optimise its processing power. Nissl staining reveals nine distinct layers to the visual (striate)

cortex, although these are grouped into a six layer scheme to maintain Brodmann’s convention (Bear

et al., 2007). Many intracortical circuits exist, and different layers output to various areas of the brain.

Cytochrome oxidase blobs were first identified by Wong-Riley in 1979, and were initially reported to

be colour, but not orientation selective (Livingstone and Hubel, 1984; Ts’O and Gilbert, 1988), as part

of a stream completely distinct from the highly orientation and spatial frequency tuned interblob

stream (Livingstone and Hubel, 1988). However, a more recent demonstration of a far more gradual

variation in neuronal properties, as distance from blob centre increases, suggests that this

dichotomous view may not be completely accurate (Edwards et al., 1995).

Additional key properties of the mammalian striate cortex, that add depth to the level of visual

processing attainable, are well described in the works of Hubel and Wiesel. Such characteristics

include the receptive field properties of simple, complex and hypercomplex cells, which achieve

summative orientation, movement and end-stopping selectivity, respectively (Hubel and Wiesel, 1965;

Hubel, 1995). They were also the first to demonstrate the presence of ocular dominance columns,

and therefore how the respective inputs to the striate cortex, of the left and right eye, are segregated

(Hubel and Wiesel, 1974). Final outputs of the primary visual cortex feed other cortical areas, the

pons and superior colliculus, or may feedback to the LGN.

Due to the small size, and relative simplicity of Drosophila’s nervous system, its optic lobes are not

nearly as extensive as the processing architecture seen in mammals. The visual system behind the

compound eye of the fly is formed of three main consecutive layers of neuropil, termed the lamina,

medulla and lobular complex. The lobular complex itself is formed of two parts; the lobula and lobula

plate, and multiple chiasms exist to connect the various neuropil. The numbers of columns in each

neuropil, easily visible with silver staining, correspond to the number of ommatidia in that compound

eye, and are referred to as cartridges (Fischbach and Dittrich, 1989). Cells in the optic lobe of

Drosophila can be classified as either columnar, or tangential. Columnar elements are those that

contribute to the formation of retinotopic maps in the various neuropil of the optic lobes, while

tangential cells are those oriented perpendicular to the cartridges.

The lamina is the most peripheral layer of the fly’s optic lobe, and exhibits possibly the most obvious

cartridge structure. However, despite the fact that the number of visual cartridges parallels the

number of ommatidia, each cartridge in fact receives input form photoreceptors of six adjacent

ommatidia. This is due to the principle of neural superposition stating that each of the peripheral

photoreceptors of an ommatidium (R1-6) has a slightly different optical axis to the others of that

ommatidium (Kirschfeld, 1967). Thus, the six photoreceptor cells that input into each cartridge of the

lamina actually originate in six adjacent ommatidia (Braitenberg, 1967), but do share optical axes.

Each cartridge of the lamina contains 12 different types of cell (Meinertzhagen and O’Neil, 1991). 11

of these are narrow field: one class of R1-6 receptor terminal, five monopolar (L1-5) and three

centrifugal cell types (C2, C3 and T1), as well as the axons of photoreceptors R7 and 8, which do not

synapse in this layer (Zhu, 2013). The twelfth type is a wide field intrinsic or amacrine cell

(Meinetzhagen and O’neil, 1991). The fibres connecting the lamina and medulla cross each other,

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such that the posterior medulla receives innervation from the anterior lamina, while subserving the

anterior visual field (Fischbach and Dittrich, 1989). This crossing forms the external chiasm.

The R7 and 8 cells project through the lamina to ramify in one or many of the 10 distinct layers of the

medulla (Zhu, 2013). Each cartridge in this layer of neuropil contains processes of approximately 60

different neurons (Takemura et al., 2008). Research using deoxyglucose labelling has shown that the

layers of the medulla may undertake differing functions (Buchner et al., 1984), supporting the idea

that insects may separate colour processing and achromatic movement detection in a similar way to

mammals (Bausenwein et al., 1992). Within the medulla exist three major visual pathways, with

proposed differing functions. The first of these pathways (L1) passes from layer 10 of the medulla to

the lobula plate, with indirect input from R1-6 photoreceptors via L1 lamina monopolar cells in the M1

and M5 layers of the medulla (Bausenwein et al, 1992). The second, L2 pathway, joins medulla layer

M9 with the shallower layers of the lobula, with R1-6 input via L2 monopolar cells. The implication of

these separate streams in motion detection arises from their meeting the requirements of input

channels to elementary motion detectors (Reichardt and Possio, 1976), and from their matching of the

spectral sensitivities found in motion sensitive lobula plate neurons (Kaiser, 1975). The two

contributing divisions of the third pathway converge in layer M8 of the medulla and connect to the

deep layers of the lobula and cumulatively receive input from cells of all spectral sensitivities

(Bausenwein et al., 1992). As such, it is suggested that M8 may be the site for trichromatic integration

in Drosophila (Straufeld and Lee, 1991).

Finally, the medulla connects to the lobula complex through the internal chiasm, where the lobula and

lobula plate face one another. Studies have shown that the primary function of the lobula plate is the

processing of movement and directional information (Bausenwein et al., 1986; Geiger and Nassel,

1982). As such, it plays a crucial role in the control of compensatory motions when the fly is moving

(Hausen and Egelhaaf, 1989). Signals are integrated here from hundreds of R1-6 pathways, and the

tangential cells of the lobula plate are generally grouped into horizontal or vertical system cells,

depending on their preferred direction of stimuli when computing the path of optic flow (Zhu, 2013).

The lobula is less well understood, but various studies have identified a small range of specialised

neurons, for example: Lobula columnar and wide field neurons (Fischbach and Dittrich, 1989), and

specialised dorsal zone cells thought to be involved in the analysis of polarised light (Fortini and

Rubin, 1990). The lobula is divided in two by the innervation from pathways two and three of the

medulla (Bausenwein, 1992). More recent studies have shown, however, that the motion detection

systems of insects and mammals, at the level of neuronal circuitry, may have been conserved far

more effectively than initially appreciated (for review, see Borst and Helmstaedter, 2015).

Comparing the roles of the eye in mammalian and insect circadian systems:

Of course, the eye also has another, highly important role in what can be termed either non-visual or

non-image-forming photoreception. Light is perhaps the most important zeitgeber for any species, and

photoreception for circadian purpose is often separated from true visual streams and is processed

differently to image-forming light information. As such, in both mammals and insects, all circadian

clocks need distinct pathways of sensory input.

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In mammals, circadian photoreception originates in the retina. However, contrary to the conventional

view of the retina, this is not confined to the traditional rod and cone photoreceptors. As far back as

the 1920s, it has been observed that rods and cones were not necessary for the generation of a

photoresponse in mammals (Keeler, 1928). Subsequent work by Provencio et al. (2000) revealed that

this ‘blind’ photosensitive ability was driven by the opsin based pigment, melanopsin. Melanopsin is a

Gq family GPCR, with an intrinsic ability to undergo photoisomerisation (Panda et al., 2005), but unlike

other photoreceptors of the mammalian retina, induces cell depolarisation upon photoisomerisation,

and so in this way resembles invertebrate opsins more than mammalian ones (Berson et al, 2002). In

their 2002 paper, Berson et al. first identified the cells responsible for circadian photoreception as

melanopsin expressing RGCs. These cells account for approximately only 1% of RGCs in the retina

(Berson, 2003), but despite this, their presence dominates the subset of the optic nerve that relays

photoentrainment to the brain (Lucas et al, 2012). This particular pathway is known as the

retinohypothalamic tract (RHT), and serves as the main route by which the brain receives light

information for circadian processing. Necessity of the RHT has been confirmed by intrinsically

photosensitive retinal ganglion cell (ipRGC) lesion studies, in which photoentrainment is lost (Guler et

al., 2008). The target of the RHT is the central circadian oscillator of the mammalian brain; the

suprachiasmatic nucleus (SCN), which coordinates all circadian variations in physiology.

Moreover though, ipRGCs do not operate as part of the circadian system on a stand-alone basis. Far

from merely transmitting the light signals they detect, studies have shown that these cells receive

additional input from rod and cone pathways as well (Belenky et al., 2003; Dumitrescu, 2009). It has

been proposed that all three types of photosensitive cell in the mammalian retina may contribute

information to the SCN at different levels of irradiance, and so code a full range of circadian

information. Rods are functional in this way at low light levels, while melanopsin expressing cells relay

information at greater intensities (Lucas et al, 2012). The role of cones in the circadian system is less

well understood, but they are thought to respond to rapid changes in irradiance, that would exceed

the temporal sensitivity of rod cells and ipRGCs to changes in light intensity (Lucas et al, 2012).

However, the circadian system may also make use of the chromatic properties of these cells. The

spectral composition of light is known to vary a great deal at dawn and dusk, and mammals may

exploit this through the use of cone cells. In fact, it has been shown that certain neurons in the SCN of

mice show colour-specific responses (Walmsley et al, 2015). In this way a chromatic, photosensitive

visual pathway may add another dimension to the information available for integration by the SCN.

Despite glaring differences in gross and neuronal anatomy of insects and mammals, the fundamental

negative, auto-regulatory nature of the circadian clock mechanism is well conserved right up to the

molecules involved (Stanewsky, 2003). A wealth of Drosophila mutants has contributed greatly to

chronobiology, beginning in 1971, when the first screen for rhythmic mutants was carried out

(Konopka and Benzer, 1971). The subsequent identification of Period (per) gene mutants led to the

identification if this and many other integral clock genes (for review: Stanewski, 2002).

The actual facilitation of photoentrainment in the fly is primarily mediated by cryptochrome, or CRY.

Encoded by the cry gene, this blue light sensitive protein was identified as a contributor to circadian

photoreception on the fly by Emery et al. (1998). CRY interacts with the timeless gene (tim) in a light

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dependent manner, causing the degradation of its gene product, TIM (Ceriani et al., 1999) and the

subsequent degradation of the PER protein to produce circadian entrainment. Photosensation in this

way produces molecular oscillations in response to light-dark cycles.

However, cryb mutant flies, which lack functional cryptochrome (Stanewsky et al., 1998) still show

entrainment in most neurons (Helfich-Forster, 2002), suggesting the presence of a CRY-independent

pathway. Opsin based photopigments have been implicated by vitamin-A depletion studies (Ohata et

al, 1998). Such photoreception may be rooted in the Hofbauer-Buchner (H-B) eyelets, residual organs

that stem from the larval eye, with a circadian sensitivity peak at approximately 480 nm (Helfrich-

Forster et al., 2002). Alternatively, cryptochrome independent circadian photoreception may occur

directly through the compound eye, via contact with a portion of the fly’s central oscillator neurons on

the surface of the optic medulla. This contact may be direct, via photoreceptors R7 and 8, or indirect

via R1-6 and a selection of laminar monopolar cells (Meinertzhagen and O’Neil, 1991). The circadian

sensitivity of the compound eyes covers an approximate range of 350-650 nm, with the CRY peak

lying outside this spread, at 420 nm (Helfrich-Forster, 2002).

In the same way that the clock cells with endogenous pacemaker activity are localised in the

mammalian SCN, Drosophila also possess a small number of neurons in the brain that show

functional circadian oscillation (Helfich-Forster, 2003). The rhythms of these central oscillator neurons

are essential for the generation of sleep-wake cycles and locomotor rhythms in the fly. Within each

hemisphere of the fly brain, there exist two groups of central oscillator neurons, termed the dorsal

(DN) or lateral (LN) neurons for their relative positions. Each of these populations can then be

subdivided several times. Of these cells, the small ventrolateral (s-LNv) neurons and the group 1 and

2 dorsal neurons begin to express oscillator activity during embryogenesis, before initial development

of the other groups (Houl et al., 2008). The LNs can be viewed as the true master circadian oscillators

in the brain of the fly for a number of reasons. Firstly, they have been shown to secrete the

neuropeptide pigment-dispersing factor (PDF), which serves as the main signal to synchronise the

central oscillator neurons (Shafer et al., 2008). Additionally, it has been shown, with the aid of disco

mutants, that it is the specific projection from the s-LNv neurons to the DN group that is essential for

rhythmic sleep-wake and locomotor behaviour (Helfrich-Forster, 1998).

Conclusion:

Research suggests that the last common ancestor of human and Drosophila lived approximately 800

million years ago (Danchin and Pontarotti, 2004). But despite this huge swathe of evolutionary time

separating the two species, it’s clear that many of the basic mechanisms used in visual perception are

well conserved. From opsin-chromophore pairing through to the use of Hassentein-Reichardt motion

detection systems, there is much commonality, and it is this that has led to the great progress brought

about by work in Drosophila. Our visual system bears almost unparalleled levels of complexity and yet

our understanding of it is incredibly extensive. Imparting the importance of using model systems,

including but not limited to the fruit fly, to the next generation of scientists will be fundamental in

maintaining such a wide breadth of knowledge. As such, it can be concluded that promoting the

immense value of Drosophila as a model system, through education and public awareness, will be

crucial to further unravelling the secrets of, not only the visual system, but our entire nervous system.

11

Project Proposal:

As summarised in the final paragraph of the above review, the fruit fly Drosophila as a model

organism is an immensely powerful tool within scientific research. The real world applications of

Drosophila research range through the entire spectrum of biological sciences, and are rooted in the

simplicity of this organism, and how easily we are able to genetically manipulate it. However, the

critical role of such model systems is scarcely emphasised with any great fervour until one reaches

higher education. I for one remember being surprised in my first year by how much neuroscience, that

I thought would be specific to the human brain and nervous system, was actually based on the fly.

With this in mind, the primary aim of my project will be to attempt to engage younger, pre-university

students in the dynamic way that the fruit fly model can be used to explain, and investigate complex

neuroscientific phenomena, to which they are likely to attribute far more salience than to the fly itself.

The desired effect of such an outreach resource will be to get younger minds thinking about how their

own questions about in depth scientific principles can be addressed using far simpler studying

techniques than they originally imagined. Additionally, this project will also aim to engrain, in potential

young scientists, the usefulness of the ‘bottom up’ approach so widely used in research involving

living organisms.

For my educational project, I propose the development of a resource that will have a wide range of

uses. For example, a demonstration that could be used to target both key stage three and key stage

four pupils. Ideally, this would be applicable to either a classroom setting, as part of an extracurricular

activity, or in a public environment, such as a science fair, where friendly engagement may be used to

demonstrate and raise awareness of the subject matter at hand. To reach these audiences, I hope to

utilise some of the existing links between the university and organisations that may act as outlets for

science education/ outreach programmes. These may include one or numerous schools that

outsource some extracurricular teaching to the Manchester Fly Facility, or institutions such as the

Manchester Museum, where events such as the Manchester Science Fair often take place.

The resources I intend to develop will include a practical demonstration of some sort, which will be

used to engage users, along with accompanying material that can be presented, either as part of the

active resource, or as an additional forum. This may include; slides, a worksheet, animation, video or

podcast that can be used in conjugate with the main resource, or as supporting material presented

separately. Examples where this may be useful might be a lecture style forum where pupils are given

background information before a practical lesson, or for people who take interest in a science fair

stand to review in their own time. I fell that supporting material will be crucial to reinforcing the

messages that outreach programmes help to convey.

For the main, interactive resource of this project, I suggest a quick and easy, ‘bitesize’ experiment that

uses Drosophila to exhibit a neuroscience based concept that would otherwise unlikely be accessible

in anything other than textbook form. For example, and to lead on from the research described in the

above review, a simple spectral preference assay, similar to those described in Gao et al. (2008) or

Yamaguchi et al. (2010).

Apparatus for such an experiment should be relatively easy and inexpensive to procure. Materials are

likely to include but not be limited to; opaque ‘Y’ or ‘T’ mazes in which the fruit flies will exhibit their

12

spectral preference behaviours, U.V., green and blue lights, with accompanying appropriate band

pass filters, flacon tubes for Drosophila transport and the flies themselves. Naturally, various

populations of Drosophila will be required, with an array of mutations that will generate a range of

spectral preferences. Such mutations might include sevenless, rdgB, ninaE, rh1/3/4/5/6/ (Yamaguchi

et al, 2010). Decisions regarding the specific strains to be used will have to be based on future

correspondence with the Manchester Fly Facility and University of Manchester laboratories.

By keeping the underlying scientific basis for the project well within the umbrella heading of ‘vision’, I

feel that benefits of the body of research already examined for the purpose of my literature review can

be maximised. When presented at an appropriate level for key stage three and four pupils, the visual

system of the fly has many merits as an educational topic. For example; it is an excellent way of

illustrating concepts at the core of basic neuroscience (synapses, neuronal pathways, action

potentials, neurotransmitters etc.) and it can be elegantly linked to genetics (so further used to

exemplify the practicality of Drosophila). Maintaining a tone of comparison between insects and

mammals, as in the literature review, may also help drive the relevance of the material home to users.

The results an assay could be used in a variety of ways, depending on the context of the

demonstration. They could be input live into a cumulative Log graph if used in a lesson, or put on the

internet (for example on the Droso4schools website) where individuals can access them and examine

the effects of certain mutations on the nervous system. Alternatively, analysis of results could serve

as an additional assignment for pupils, either in a homework format or as an extra/ follow on activity.

Evaluation of the project will be highly important. Evaluation of the success of the resources produced

should take multiple forms. These will include some sort of formative test, or assessment of users

(hopefully) improved knowledge of the subject matter, as well as some subjective feedback that will

help younger people describe how interesting, insightful and relevant they feel the resources have

been. I also intend to afford participants, and possibly parents or teachers as well, the opportunity to

feedback over a longer period. The documentation of interest levels and participation in similar

science based activities or educational opportunities in the future will undoubtedly add another useful

dimension to the evaluation of the project.

Obviously, these ideas are still rather immature and by no means a finalised project plan. However, I

hope it has become clear that I intend to develop a project that will have varied, and widespread

positive effects on the younger people who use it. The value and relevance of the Drosophila model

system can never be stressed enough, and hopefully this project will make headway into introducing

that concept Drosophila based research to budding young scientists, well before it is presented to

them in a university environment. Using a variety of media to create new educational aids, as well as

building on some of those that the University of Manchester, the Droso4schools website and the

Manchester Fly Facility, I hope to bring neuroscience into view for many who may not yet have seen

its beauty.

13

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