Montagem de Retina

8/13/2019 Montagem de Retina

1/19

Fax +41 61 306 12 34

E-Mail [email protected]

Original Paper

Brain Behav Evol

DOI: 10.1159/000332802

The Retinal Wholemount Technique:A Window to Understanding the Brain andBehaviour

Jeremy F.P. Ullmanna Bret A. Moorec Shelby E. Templea

Esteban Fernndez-Juricicc Shaun P. Collina, b

aSchool of Biomedical Sciences, The University of Queensland, Brisbane, Qld. , and bSchool of Animal Biology and

the UWA Oceans Institute, The University of Western Australia, Perth, W.A., Australia; cDepartment of Biological

Sciences, Purdue University, West Lafayette, Ind., USA

cies-specific differences encountered when examining a

range of vertebrate taxa (fishes, reptiles, birds and mam-

mals). This broad comparative approach will enable future

studies to overcome technical difficulties, thus permitting

larger conceptual questions to be posed regarding the di-

versity of visual tasks across phylogenetic boundaries.

Copyright 2011 S. Karger AG, Basel

Introduction

The retina is one of the most easily accessible parts ofthe central nervous system in vertebrates. This thin sheetof nervous tissue that lines the back of the eye processesthe optical image of the visual field, transforming lightenergy into electrical signals (i.e. phototransduction) toform a neural image interpretable by the rest of the brain.Physically isolated from the rest of the central nervoussystem, it provides a window through which to examine

how each species perceives their environment, a conduitfor visual information that is filtered and processed by thebrain before being used in the initiation of behaviours.

This year marks 30 years since the publication of TheWholemount Handbook. A Guide to the Preparation andAnalysis of Retinal Wholemountsby Stone [1981]. In hishandbook, Stone [1981] presented the wholemount tech-nique and numerous methods for visualizing ganglion

Key Words

Vision Ganglion cell Photoreceptor Oil droplet Visual

acuity Neuroethology Topography Behavioural ecology

Abstract

The accessibility of the vertebrate retina has provided the

opportunity to assess various parameters of the visual abili-ties of a range of species. This thin but complex extension of

the brain achieves a large proportion of the necessary visual

processing of an optical image before information is deliv-

ered to the brain as neural impulses. Studies of the retina as

a wholemount or a flattened sheet of neural tissue are abun-

dant due to the large amount of information that can be an-

alysed, as follows: the level of summation or convergence;

the coverage, stratification and potential sites of synaptic

connections; the spatial resolving power; the arrangement

of neuronal arrays or mosaics; electrophysiological access

for the recording of responses to visual stimuli; the spatial

arrangement of cell dendritic fields; location of retinal blindspots (optic nerve, falciform process and pecten); topo-

graphic differences in retinal cell sampling; spectral filters,

and reflective structures. The present study examines all as-

pects of the wholemount technique, including enucleation,

fixation, retinal extraction, flattening, staining, visualization

of labelled cells and stereological mapping of cell density.

Uniquely, it highlights the crucial technical and often spe-

Received: June 3, 2011

Returned for revision: July 15, 2011

Accepted after revision: August 31, 2011

Published online: December 6, 2011

Jeremy F.P. UllmannSchool of Biomedical SciencesThe University of Queensland

Brisbane, QLD 4072 (Australia)Tel. +61 7 3409 9058, E-Mail j.ullmann @ uq.edu.au

2011 S. Karger AG, Basel00068977/11/00000000$38.00/0

Accessible online at:www.karger.com/bbe


2/19

Ullmann /Moore /Temple /Fernndez-Juricic /Collin

Brain Behav Evol2

cells predominantly but also photoreceptor cells in the catand turtle. Various additional staining techniques usingcresyl violet, methylene blue, catecholamine fluorescence,Golgi and reduced silver impregnation methods were alsoincluded in the text, as well as the cellular preservationand visualization of a stable reaction product following

incorporation of various retrograde tracers such as horse-radish peroxidase into the optic nerve. Stones [1981] tech-nical monograph laid the foundation for what was to be-come a vital tool in the neurosciences, one which has tak-en full advantage of the plethora of fluorescent labels nowavailable to intracellularly stain neurons.

The retinal wholemount technique is a useful methodof assessing the structure, arrangement and sampling ofretinal neurons in vertebrate eyes. Numerous visual pa-rameters can be determined with the wholemount tech-nique, including:

1 The degree of summation or convergence of informa-tion between photoreceptor and ganglion cell popula-tions, by examining cell distributions on the same ret-ina and at each of the retinal loci and then construct-ing topographic maps to identify the levels ofsummation across the retina [Litherland and Collin,2008].

2 Coverage, stratification and potential sites of synapticconnections with other retinal neurons following theinjection of intracellular dyes [Dacey and Peterson,1992; Vaney et al., 1989] and either in vitro (living andsuperfused with physiological Ringers solution) or

retrograde labelling from the optic nerve in vivo [Col-lin, 1989b].

3 Spatial resolving power of the optical (using the pho-toreceptor spacing) and neural (using the ganglion cellspacing) image when combined with measures of focallength and/or posterior nodal distance (PND) [Collinand Pettigrew, 1989; Pettigrew et al., 1988]. Estimatingspatial resolving power across species has been essen-tial to study the evolution of visual acuity in specieswith different life histories and different activity peri-ods [Garamszegi et al., 2002].

4 Spatial arrangement of neuronal arrays (regular and

irregular mosaics) with increasing eccentricity mosteasily visualized for photoreceptors using Nomarskior differential interference contrast optics [Collin etal., 2004; Litherland and Collin, 2008] in order to as-sess chromatic sampling and motion sensitivity.

5 Intracellular electrophysiological recording of re-sponses to visual stimuli in superfused wholemountedretinas incubated in Ringers solution and maintained

at optimal recording temperature [Wong et al., 2005].Multiple simultaneous recording electrodes are cur-rently being used to record waves of activity acrossthe retina [Segev et al., 2007; Wong et al., 1993].

6 Confocal measurements of neurons and their dendrit-ic fields using both two-dimensional and three-dimen-

sional (z-series) analysis [Koch et al., 2010], enablingmore accurate assessment of retinal coverage and therelationship/connectivity with other neuron types.

7 Topographic arrangement and density of natural fea-tures of neurons, such as intracellular oil droplets andyellow filters [Hart et al., 1999], fluorescent taggedprobes in retinal tissue incubated for in situ hybridiza-tion and identification of visual pigment opsins [Bow-maker et al., 1997; Schiviz et al., 2008; Takechi andKawamura, 2005] and other neurons or glia pre-la-belled using immunohistochemistry, Golgi and silverstaining techniques and/or cells retrogradely labelled

using carbocyanine dyes [Thanos et al., 1992; Vidal-Sanz et al., 1988]. These parameters are used in thestudy of the evolution of colour vision and spectraltuning in vertebrates [Collin et al., 2009; Collin andTrezise, 2004; Collin and Trezise, 2006].

8 The location, size and shape of fundal landmarks suchas the avian pecten [Brach, 1977; Smith et al., 1996], theteleost falciform process [Collin and Pettigrew, 1988a],the reptilian conus [Dieterich et al., 1976], the hyalinevasculature [Collin, 1989a; Collin and Pettigrew,1988a; Dunlop et al., 1997] and the location of the op-tic nerve head [Frank and Goldberg, 1983]. These fea-

tures define the position of blind spots and the influ-ences of a vascular blood supply on acute vision.

9 Topographic differences in the concentration of re-flective structures (tapetal material) underlying theretina, which can be used as an indicator of retinal re-gions of enhanced sensitivity in species occupying lowlight environments [Ollivier et al., 2004; Somiya, 1980;Takei and Somiya, 2002].

10 Peak spectral sensitivities of multiple photoreceptorsarranged into two-dimensional arrays simultaneously[Levine et al., 1979]. This could enable efficient sam-pling across large retinal regions, in order to ascertain

the presence of intra-retinal variability [Levine et al.,1979; Temple, 2011; Temple et al., 2010].

Examination of the retina using the retinal whole-mount technique has enabled us to investigate the visualcapabilities of over 300 species of vertebrates. Many pa-pers have been written, with over 150 published in Brain,Behavior and Evolutionjust in the past decade. The large


3/19

The Retinal Wholemount Technique Brain Behav Evol 3

amount of information produced using the techniqueand its utility in facilitating comparative studies has beenhighlighted by the creation of two resources. The first isa public archive of retinal topography maps (see http://www.retinalmaps.com.au/) which, together with rele-vant information about eye size, retinal cell density, reti-

nal orientation, cell number, spatial resolving power andthe type of retinal specialization, provides a searchableonline resource of over 1,200 topography maps for 300species across all vertebrate taxa, and the second is an in-ternational working group on the Evolutionary shifts invertebrate visual ecology and visual morphology sup-ported by the National Evolutionary Synthesis Centrein the USA. A large number of the studies this workinggroup is conducting are based on papers that used theretinal wholemount technique.

The present study includes (1) a detailed protocol forthe retinal wholemount technique, including numerous

technical refinements that we have developed for specifictaxa, (2) issues relevant to the use of theoretical calcula-tions based on data extracted using this technique, e.g. thedifferentiation of cell types and the calculation of spatialresolving power, and (3) the breadth and utility of the ret-inal wholemount technique and its major applications, notonly to neuroscience but also other disciplines (e.g. visualecology, visual physiology, behavioural ecology). We pro-vide information that will hopefully improve method-ological standardization of the wholemount techniquewhen used across a diversity of phyla. This should in turnfacilitate comparative research and meta-analyses, which

will enhance our understanding of the plasticity, adapta-tion and evolution of the visual system of vertebrates.

Materials and Methods

The comprehensive protocol detailed in this paper is the prod-uct of over 20 years of research. During this time, we have exam-ined a multitude of species from the two largest vertebrate groups,the birds and fishes. To supplement our technical knowledge inthese classes of vertebrates, we have also performed an extensiveliterature review. The result is a complete methodology that rang-es from enucleation of the eye to the creation of topographic maps.

For many of the steps, we provide different options depending onthe organisms being examined. For example, to open the eyeballin birds we recommend hemisecting the retina, while for teleostswe suggest removing the cornea (the transparent window that cov-ers the iris, pupil and anterior chamber). In addition, we describeprocedures for the following: the removal of the vitreous humour(gel-like liquid that fills the cavity of the eye) by enzymatic diges-tion, which is vital for analysis of deep-sea teleosts; bleaching ofthe pigmented cell layer found between the retina and the choroidcalled the retinal pigmented epithelium (RPE), which is helpful in

taxa/species in which the RPE adheres closely to the photorecep-tors (e.g. birds and some teleosts); and visualizing photoreceptorsand ganglion cells in the same retina, which permits the calcula-tion of summation ratios within a single retina. For the first-timeuser of the wholemount technique, we have included a reagent andequipment list, a reagent setup, a basic cresyl violet staining pro-tocol (table 1), a glossary and an extensive bibliography. Moreover,we have provided an example investigation of the archerfish Tox-otes chatareus to demonstrate the utility of the protocol.

Reagents

The following reagents are required: 0.1 Mphosphate buffer (see Reagent Setup below) 4% paraformaldehyde (see Reagent Setup) formaldehyde nail varnish 100% glycerol Ames medium (Sigma-Aldrich Pty. Ltd., Sydney, Australia) Gatenbys solution (see Reagent Setup) bleaching solution (see Reagent Setup) ethanol

Table 1.Basic cresyl violet staining protocol

Stage Staining tray Time Purpose

1 Histoclear 10 min clearing agent2 Histoclear 10 min clearing agent3 100% ethanol 2 min defatting1

4 100% ethanol 2 min defatting15 90% ethanol 2 min rehydration6 70% ethanol 2 min rehydration7 20% ethanol 2 min rehydration8 distilled water 2 min rehydration9 0.1% cresyl violet2 20 min3 staining

10 distilled water4 quick rinse rehydration11 20% ethanol 30 s dehydration12 70% ethanol 30 s dehydration13 90% ethanol 30 s dehydration14 differentiation solution 1 min differentiation15 100% ethanol 1 min dehydration16 100% ethanol 1 min dehydration17 100% xylene or Histoclear 2 min5 clearing agent

18 100% xylene or Histoclear 2 min

5

clearing agent1 This stage can be left for up to 24 h.2Higher concentrations can be used for a deeper, darker stain.3 Staining should be monitored using a compound micro-

scope. Alter staining time appropriately, keeping in mind thatsome fading may occur later on during dehydration. Longerstaining times will be required for visualization of ganglion cellslying within the inner nuclear layer.

4A preliminary analysis of the degree of staining and back-ground clarity can be performed at this step by holding the retinato the light.

5This time can be extended if necessary. Xylene and Histo-clear are miscible with Depex, used for cover-slipping.


4/19


Brain Behav Evol4

xylene Histoclear 0.1% cresyl violet in acetate buffer (see Reagent Setup) differentiation solution (see Reagent Setup) Depex mounting media (Grale Scientific Pty. Ltd., Vic., Aus-

tralia) Fluoromount aqueous mounting media (Sigma-Aldrich)

sodium azide nitric acid hydrochloric acid hyaluronidase (Sigma-Aldrich).

EquipmentThe necessary equipment is listed below: digital callipers 2 dissecting forceps No. 5 (Fine Science Tools Inc., Calif., USA) iridectomy scissors (Fine Science Tools) scalpel surgical blades No. 11 razor blade 2 camel hair (natural animal hair) paintbrushes (can be pur-

chased from art supply stores) slides (size depends on size of retina) cover slips (No. 1 thickness) Bunsen burner specimen block Kim wipes or tissue paper black Millipore filter paper (HABP02500, Millipore Pty. Ltd.,

Vic., Australia) glass staining dish 100-ml glass beaker heating tray graph paper dissecting microscope compound microscope with a stage with x- and y-axis rulers eyepiece graticule with a 10!10 grid

calibration slide digital camera compound microscope fitted with an X, Y and Z motorized

stage Stereo Investigator software (MicroBrightField Inc., Vt., USA).

Reagent SetupStandard 0.1 Mphosphate buffer at pH 7.4 is suitable for this

protocol. Store at 4 C and discard if precipitate forms.Paraformaldehyde (4%) is the fixative commonly used, pre-

pared in 0.1 Mphosphate buffer.An acid mixture is used to clean slides and the cover glass.

Prepare by mixing 2 parts nitric acid to 1 part hydrochloric acidin a fume hood.

Gatenbys solution is used to sub the slides. Prepare by mixingSolution 1 into Solution 2, as follows: Solution 1: mix 2 ml of distilled water and 0.1 g of chromium

potassium sulphate (chrome alum). Solution 2: heat 1.5 g of gelatine in 7 ml of glacial acetic acid

until the gelatine dissolves.Bleaching solution is prepared by mixing 90 ml of 0.1 Mphos-

phate buffer (pH 7.4) and 10 ml of 30% hydrogen peroxide. Adjustthe pH to 11.95 using 1.0 Mpotassium hydroxide (this wil l requireapprox. 80 ml).

Cresyl violet (0.1%, pH 3.7) in acetate buffer is prepared by mix-ing 0.4 g of cresyl violet, 10 ml of glacial acetic acid, 10 ml of 1.0 Msodium acetate and 380 ml of distilled water. Filter before use.

Differentiation solution is prepared by mixing 900 ml of 95%ethanol and 1 ml of glacial acetic acid.

Results

In the following we provide a detailed series of steps tosuccessfully use the wholemount technique. Note that allthe examples provided in this protocol were performedunder the ethical guidelines of our respective researchinstitutions.

Orientation and Enucleation of the EyeDark-adapt the animal for at least 2 h prior to dissec-

tion. In most teleost species, this facilitates the removalof the RPE. However, in other species, such as birds, we

recommend timing the dissection to match the circadianrhythm of the animal with the time-period in which theRPE adheres less strongly working best (i.e. dissectingavian eyes in the evening tends to work better than in themorning). Prior to removing the eye, note any externalcharacteristics that can be used later for orientating theeye (e.g. aphakic spaces, an area of the pupil throughwhich light can reach the retina without first passingthrough the lens, or differences in pigmentation betweenthe dorsal and ventral surfaces of the iris, a circular struc-ture made up of two cellular layers, stroma and pigment-ed epithelial cells, which is responsible for controlling the

diameter of the pupil; fig. 1). If none exist, it may be nec-essary to mark the eye by placing a small cut in the retinaor alternatively mark the iris using a portable cautery.Gently rotate the eye forward and cut the conjunctiva(transparent mucous membrane that extends from thesurrounding epidermis over the cornea or clear windowof the eye). Gently pull the eye out of the orbit and cutaway the adipose tissue, extraocular muscles and opticnerve. Be sure to leave a section of the optic nerve at-tached to the eye. Once the eye has been removed, cutaway extraneous tissue, such as the extraocular musclesand connective tissue attached to the back of the eyecup.

Measure the corneal diameter just outside the scleroticring/bones (rings of bony plates inside of the sclera whichare present in many reptiles and birds) or where the cor-nea meets the sclera (the fibrous outer tissue covering theentire eye except for the cornea), as well as the transverseand axial lengths of the eye (fig. 2). Measurements shouldbe taken at the point where the callipers make contactwith the eye, but do not squeeze or depress the eye.


5/19


6/19


Brain Behav Evol6

the scalpel blade parallel to the cornea to reduce the riskof damage to the retina (fig. 3a, b). If necessary, aim thescalpel blade slightly upwards. Create an incision at theborder of the cornea and sclera (the limbus) large enoughfor a pair of iridectomy scissors to be inserted. Cut alongthe edge of the cornea and iris with the iridectomy scis-sors until the corneal surface of the eye has been removed.If using a razor blade to hemisect the eye, first stabilizethe eye by gently squeezing the eye with forceps. Apply aslight downward pressure with one arm of the forceps to

the top of the cornea, which will help stabilize the eye(fig. 3c). Make sure that the pupil is perpendicular to thetable to ensure that the blade cuts straight. Locate the oraserrata and begin by slicing rather than pushing, using adelicate, long, slow sawing motion to make the initial cut.Follow by applying downward pressure while continuingto saw until the eye is completely hemisected. Cutting be-low (towards the back of the eye) the ora serrata will dam-

age the retina and result in data loss, while placing thepuncture too far above will make retinal extraction moredifficult.

Once the cornea has been removed or the eye hemi-sected, examine the retina for physical landmarks that canbe used to indicate orientation. Note whether it is the leftor right eye and the orientation of dorsal, ventral, temporaland nasal poles. In mammals, the area centralis (a concen-tric increase in cell density towards the central area ofacute vision) usually lies temporal to the optic disc (the

area where ganglion cells exit the eye to make up the opticnerve; it contains no rods or cones and is therefore knownas the blind spot) and therefore distinguishes the nasaland temporal sides of the retina; in birds, the pecten (acomb-like structure made up of blood vessels found in thevitreous body but part of the choroid) can be used to ori-entate the eye, and in some teleosts the position of the op-tic nerve head and/or the falciform process (a projection

a b c

C

olorversiona

vailable

online

Fig. 2.Eye measurements. aTransverse length. bAxial length. cCorneal diameter.

a b c

Fig. 3.Opening the eyeball. aDorsal perspective of puncturing the eye using a scalpel. bLateral perspective of scalpel technique.cHemisecting the eye using a razor blade.

Colorversiona

vailab

le

online


7/19


8/19


9/19


Once the retina has been completely removed from theeyecup, grasp the optic nerve with one set of forceps andwith another pair of forceps gently peel and, if necessary,cut away the choroid/choriocapillaris, the underlyingRPE and, if present, any tapetal tissue (the layer of tissuefound behind or within the retina that reflects visible

light back through the retina). Begin peeling from theoptic nerve and work towards the edge of the retina. If itis not possible to peel away the RPE, use an animal hairpaintbrush to gently brush the RPE away from the backof the retina. Care should be taken when brushing awaythe RPE as it is possible to remove the photoreceptor in-ner/outer segments at the same time. Note that the RPEis denser than the retina, so with the retina submerged,pieces of RPE will sink to the bottom of the container. Itis also brittle and can break into tiny granular pieces, soonce removed from the retina, place discarded pieces ofRPE away from the retina or wipe them from dissection

tools with tissue paper. If layers of the retina are slough-ing off while removing the RPE, the retina is under-fixed.If this occurs, it may be possible to place the retina backinto the fixative. However, most likely a new eye will haveto be dissected. Note that it may not be possible to com-pletely remove the RPE, and therefore bleaching (see be-low) will then be required in order to produce a transpar-ent retina suitable for staining and visualization withtransmitted light. In larger species, the RPE is often quitethick and must be removed physically as the bleachingprocess will be less effective. Bleaching cannot be used ifoil droplet distribution will be mapped, and thus the RPE

must be removed physically with a brush. Bleaching willnot be necessary if visualizing fluorescently labelled reti-nal neurons in wholemount. Finally, cut away the re-maining optic nerve and sclera with a scalpel.

Retinal FixationPlace the retina/eyecup in 4% paraformaldehyde in

0.1 Mphosphate buffer to fix the retina. Make sure thereis enough fixative to completely submerge the entire ret-ina/eyecup. This should equate to roughly 5 t imes the vol-ume of the eye. The time of fixation varies depending onthe size and thickness of the retina. A 1-cm-diameter te-

leost eye should be fixed for between 60 and 120 min,while a bird eye of the same size may safely be fixed forbetween 12 and 24 h and the retina will still be success-fully wholemounted. Remove the retina/eyecup from thefixative solution by grasping the retina/eyecup by the op-tic nerve. Then wash it in 0.1 Mphosphate buffer (1 min)to remove excess fixative (repeat 3 times). If the opticnerve has been cut off the retina, it is safer to remove the

fixative from the container and then rinse the tissue inthe original container by replacing the liquid and swirl-ing the container with each rinse. Once the retina is fixed,samples can be stored in 0.1 Mphosphate buffer (pH 7.4)at 4 C for several weeks; however, if leaving the retinaefor a longer time period, 0.010.05% sodium azide should

be added to inhibit bacterial growth [Lichstein and Soule,1944].

Bleaching the RetinaWash the retina in fresh 0.1 Mphosphate buffer and

then replace the buffer with bleaching solution (see Re-agent Setup). Completely submerge the retina; this shouldequate to at least 5 times the volume of the eye, dependingon the volume of the container used. The length of timerequired to bleach the RPE is dependent on the thicknessand amount of RPE remaining. During bleaching, theRPE will fade in colour from black to dark brown to light

brown to yellow/clear (fig. 5). Additionally, bubbles willform along the edges of the retina. Be aware that duringthe bleaching process, the retina may change position andorientation. We suggest taking digital pictures through-out the process to assist with maintaining the orientationof the retina. In addition, leaving the retina in the bleach-ing solution too long will cause it to become progressive-ly softer, eventually making it too delicate to handle. Af-ter the bleaching process is complete, replace the bleach-ing solution with 0.1 Mphosphate buffer and wash/rinsethe retina twice for 1 min each time.

Wholemounting the RetinaA clean slide and cover slip are required for whole-

mounting the retina for photoreceptor visualization,while a clean and (previously) gelatinized slide is requiredif wholemounting is followed by staining for ganglion cellvisualization. Clean the slides and cover glass by placingthem into nitric and hydrochloric acid solutions (see Re-agent Setup) for approximately 2 h with occasional swirl-ing. Then rinse the slides and cover glass thoroughly withwater and store in 70% ethanol. Finally, flame the slidesand cover slips in a fume hood prior to use. Gelatinizeglass slides (size dependent on the size of the retina) by

cleaning the slides and dipping them into Gatenbys solu-tion (see Reagent Setup) 3 times. Let the slides dry be-tween dips. To decrease the drying time, place the slidesinto a warm oven (37 C) or blow-dry them.

Float the retina onto either a gelatinized slide for gan-glion cell visualization or a clean (but non-gelatinized)slide for photoreceptor visualization. Rotate the retina tomatch the orientation of the eye in the orbit/skull, noting


10/19


Brain Behav Evol10

whether the eye is the left or right. To visualize photore-ceptors, the inner (vitreal) surface of the retina should bemounted face down. Place the inner retina face up forganglion cell visualization. Flatten the retina by makingradial cuts with a scalpel blade or scissors around the pe-riphery of the retina to release tension and allow it to lieflat. There are no set positions for where to best make ra-dial cuts, as all retinas are different. However, if a rip orhole is already present in the retina (due to a difficultyduring dissection), place a radial cut there or, if possible,place a radial cut along the falciform process or pecten

(when present). Try not to place cuts in regions predictedto represent a retinal specialization (e.g. region of highcell density). If the retina possesses a pecten (birds) orconus, a cone-like structure located on the optic nervehead (reptiles), remove the pecten/conus and any otherconnective tissue with forceps and iridectomy scissors.Similarly, if parts of the optic nerve head are protruding(e.g. hyoid vasculature of teleosts), remove these carefully

with scissors. This will allow the retina to lie completelyflat and adhere to the slide. Use filter paper to soak up theexcess buffer and a paintbrush to gently flatten the retina.By absorbing the excess liquid from underneath the reti-nal tissue, the retina will stick to the gelatine-coated slide.The retina may curl tightly in on itself. This often occurswhen the retina has been fixed (especially for longer pe-riods of time) after extraction and is normal. However, ifit is difficult to uncurl and flatten, the retina may havebeen over-fixed (rubbery, hard or brittle), or some of thevitreous may still be attached. If the retina has been over-

fixed, placing it back in the bleaching solution will softenit. If there is any vitreous remaining, it must be removedprior to (flattening and) effective staining. Use scissorsand forceps or filter paper to remove excess vitreous or,if difficult, use the hyaluronidase and/or collagenaseslurry as described in the section Extraction of Retina forWholemounting. Also, if the peripheral cuts do not pen-etrate enough towards the central retina, the retina will

a b

c d

C

olorversiona

vailable

online

Fig. 5.adProgressive bleaching of a reti-na. Note the appearance of air bubbles andthe change in orientation of the retina.


11/19


not lie flat. Use scissors to increase the size of the periph-eral slits.

If the flattening procedure takes too long (11020min) while mounting for ganglion cell visualization, theretina may not readily stick to the slide. This may be dueto the gelatine on the gelatinized slide being washed away.

In this case, transfer the retina to a new slide and con-tinue the procedure. Double and triple subbing of theslide may also improve retinal adhesion, especially forlarge eyes.

Visualization, Staining and Labelling of Retinal CellsPhotoreceptors and ganglion cells can be viewed inde-

pendently, on different retinas, or on the same retina ifphotoreceptors are examined before staining for gangli-on cells.

Photoreceptors

To prevent the retina from being crushed and to allowthe photoreceptors to maintain their natural orientationand density, create a spacer out of plastic, paper or tapeand place this between the slide and cover slip. The spac-er should be of a slightly greater thickness than the retinaand have a hole in the middle large enough for the flat-tened retina to sit inside. Place the spacer onto the slidecontaining the retinal wholemount and glue the spacer tothe slide using nail varnish (polish). The spacer is bestfixed to the slide before the retina is transferred to theslide.

Place a few drops of 100% glycerol onto the buffer-

soaked retina such that the final proportion of liquid onthe slide is 50% glycerol and 50% buffer. Place one edgeof the cover slip on the slide and then gently lower thecover slip onto the retina using forceps. Lowering the cov-er slip down from one side will prevent bubbles from be-ing trapped over the retina. Seal the cover slip to the plas-tic spacer with nail varnish, to reduce the risk of the ret-ina being exposed to air. If the retina dries out, thephotoreceptors will deteriorate and the slide will becomeuseless.

Observe the photoreceptor array under a compoundmicroscope. Nomarski or differential interference con-

trast optics will facilitate photoreceptor visualization andcounting. If the photoreceptors appear squashed, thespacer is likely too thin and/or there may still be vitreousattached and/or the manual handling of the retina hasbeen severe. To solve this problem, submerge the wholeslide in PBS and remove the cover slip by breaking theseal around the edge with a scalpel blade using a twistingmotion. Then gently slide the old cover slip off and re-

place the plastic spacer with a thicker spacer. Take care toreduce direct contact with the photoreceptor layer.

Some taxa have oil droplets located within the innersegment of cone photoreceptors (e.g. some birds, reptiles,amphibians, fish and monotreme and marsupial mam-mals). Since each type of oil droplet is associated with a

specific type of photoreceptor [Hart, 2002], the identifi-cation of different oil droplet types across the retina al-lows the distribution of different types of photoreceptorsto be determined. In order to visualize the oil droplets,the retina has to be fresh (no fixation, no bleaching),mounted just as one would for ganglion cell visualization(see below). Mapping the oil droplet distribution is donein a similar manner as for ganglion cells (see below); how-ever, to distinguish the different types of oil droplets, acombination of brightfield and fluorescent microscopy isnecessary (for details, see Hart [2001]).

Ganglion CellsTo visualize and assess the ganglion cell population

following assessment of the photoreceptors, slide a scal-pel blade under the cover slip and slowly twist the handleto crack the cover slip. Use forceps to carefully remove thebroken pieces of cover slip so as not to tear the underlyingretina. Soak the entire slide in phosphate buffer to removeglycerol and assist in floating the retina off the slide. Ananimal hair paintbrush may be needed to gently detachthe retina from the slide.

Float the retina (ganglion cell layer face up) onto afreshly gelatinized slide. Using a paintbrush, arrange the

retina so that the inner retina is facing up and its orienta-tion is consistent with the placement of the eyes/retina inthe skull. Use a paintbrush to flatten the retina and to re-move any air bubbles (fig. 6a). Use the paintbrush to en-sure that the retina is well attached to the gelatinized slideat its edges. Photograph the entire retina beside a calibra-tion tool (ruler or micrometer) in order to account for anyshrinkage that will occur during the drying and stainingprocesses. Dry the retina to the slide. Avoid over-dryingas the retina will not stain well and it may even crack. Thisstep can be performed using option 1 or 2 (below), de-pending on the urgency with which the sample is needed.

Option 1: place a damp (water) paper towel in an air-tight box, followed by the slide, horizontally on a stand(e.g. the lid from a specimen tube). Then slowly decreasethe moisture in the container by slightly opening its lid alittle more each day. This will allow the retina to dry slow-ly over a period of several days.

Option 2: place the slide (horizontally) in a containerwith a lid together with a small piece of tissue paper


12/19


Brain Behav Evol12

soaked in a few drops of 37% formalin (only vapoursneeded). Place a paper towel between the lid and the con-tainer to catch any condensation that forms on the lid, asdroplets of water will ruin the retina (fig. 6b). Heat at60 C for 30120 min (depending on the size/thickness ofthe retina). Turn off the heat and let it sit overnight.

Stain the retina using a 0.1% cresyl violet solution. SeeMaterials and Methods for a suggested protocol. Place afew drops of mounting media onto the retina and slowlylower a cover slip onto it from one side, to stop bubblesfrom being trapped over the retina. The cover slip shouldbe large enough to cover the retina and extend a few mil-

limetres beyond. Add mounting media around the edgesof the cover slip, if necessary, in order to prevent any airbubbles from forming. Remove any excess mounting me-dia with tissue paper. Take another photograph of the en-tire retina beside the same calibration tool and estimatethe level of shrinkage.

Observe ganglion cells under a compound micro-scope. Ganglion cells usually possess an irregularlyshaped soma with Nissl substance and a distinct nucleus,while amacrine and glial cells are either teardrop or slen-der in shape, respectively [Ehrlich, 1981]. If observing f lu-orescently labelled ganglion cells (either injected intracel-

lularly or retrogradely labelled from the optic nerve),mount as per the photoreceptors. In this case, a mountingmedium such as Fluoromount aqueous mounting medi-um (Sigma-Aldrich) can replace glycerol in order to in-hibit fading. The wholemounted retina (stained with cre-syl violet) protected with a cover slip will last for monthsto years, although the Nissl staining will fade over time.If this occurs, it is possible to remove the cover slip by

soaking in xylene for days to weeks and repeating thestaining and mounting.

Mapping Cell DistributionsThis step can be performed using either an eyepiece

graticule or stereological computer software, dependingon the equipment available.

Mapping with an Eyepiece GraticuleObserve the retina under the compound microscope

with a low-power objective to obtain the coordinates ofthe extremities of the retina (i.e. the coordinates of the

outer and inner edges of the radial cuts). Draw a basicoutline of the retina onto a piece of graph paper (fig. 7a).Use the coordinates to determine the scale for drawingthe retina onto graph paper (suggested ratio 1: 10 or 1: 5).Carefully draw a preliminary outline of the retina ontograph paper. Scan the retina in approximately 1-mmsteps (smaller or larger steps can be used depending onthe size of the retina) to confirm the coordinates of theretina outline and to add any relevant features (e.g. opticdisc, falciform process, pecten, vitreal blood vessels andfovea) to the graph paper. This retinal outline and thelines of the graph paper will be used as a grid to record

the location of each sample count.Based on the area of the retina, determine the spacing

needed between counting areas in order to obtain ap-proximately 200 sampling areas per retina and an accept-able coefficient of error (CE; see below). The higher thenumber of sampling areas, the more accurate the subse-quent topographic distribution map will be. Change to ahigher-power objective and begin counting cells within

a

b

c

Fig. 6.Preparation of a retina for counting. aFlattening the retina using animal hair paintbrushes. bRapid dry-ing of a retina using a heating tray and formalin. cA dried retina ready for cresyl violet staining.

C

olorversiona

vailable

online


13/19


the boundaries of the 10!10 graticule. To prevent dou-ble counting, only count cells that lie solely within eachsquare grid and touch only two sides (e.g. top and rightsides of the square grid). Perform a few practice counts toensure accuracy and reproducibility. Counters availableinclude mechanical counter wheels and electronic types.For calibration, note the area of the graticule field size foreach objective used for counting and determine the size

of the sampling area in square millimetres. Accuracy willbe improved if cell counts per graticule range between 20and 200 cells. Consider the potential range of cell densi-ties in the retina when selecting objective strength. It maybe necessary to examine regions of the retina to estimatecell density before commencing counting.

Count the number of cells in each sampling locationand then move the microscope stage to the next sampling

location and continue counting. Subsample the entireretina in a methodical pattern. Note that in areas of rapidchanges in cell density, the level of subsampling may haveto be increased, i.e. in high-density areas associated withareae centrales and streaks, in order to accurately defineand account for rapid topographical changes in density(fig. 7b). Occasionally, it may not be possible to count thecells in the current sampling area due to the cells being

oriented on their side, damage to the retina, inadequateremoval of the vitreous or the staining not being prop-erly timed (or appropriately adjusted for retinal thick-ness/size). If this occurs, move on to the next samplingarea and try and determine the source of error, so it canbe corrected for the next retina. In addition, accuratecounting may not be possible when the number of sub-laminae (e.g. in the ganglion cell layer) do not allow one

a cb

d e f

Fig. 7.Topographic map creation. aOutlined retina. Note that theoptic nerve head and falciform process have been excluded fromthe retina. bRetina containing sample counts. cEach numeralrepresents the number of cells counted within a counting frameof 100m2. dThe counts are converted to square millimetres. In

this example, each numeral is multiplied by 102; thus, a measure-ment of 93 cells/100 m2would be 9,300 cells/mm2. eIso-densitylines can then be constructed by interpolation between cell distri-bution counts. fCompleted topographic map, with progressivelydarker blue indicating increasing cell distributions.

C

olorversiona

vailable

online


14/19


Brain Behav Evol14

to differentiate the number of cells over multiple layerseven with fine adjustment of the fine focus of the micro-scope.

Mapping Using Stereological Computer SoftwareUse a compound microscope fitted with an X, Y and

Z motorized stage and linked to a computer running ste-reological software such as Stereo Investigator (Micro-BrightField) to digitize the retina under low power. Onthe computer, outline the limits of the retinal bordersalong the radial cuts and any internal structures such asa pecten or falciform process.

Using the stereological software, place random andsystematic counting boxes on a grid that covers the entireretina. About 200 counting points should be placed on aretina. The size of the counting box and intervals betweencounting points will be determined by the size of the ret-ina. For example, for a retina that can be covered by a 750

!750-m grid, use a 25!25-m counting box. How-ever, this may vary depending on cell densities, as countsin any given box should be between 20 and 200 cells. Toofew counts reduce the accuracy, while too many countsrequire an excessive amount of time.

After sampling the entire retina, locate areas of highercell density such as an area centralis, fovea or horizontal/visual streak and perform higher-frequency sampling.

To determine if enough counts are being performed,calculate the Schaffer CE, which indicates the precision ofthe cell number estimates [Fileta et al., 2008]. An accept-able CE can be calculated using the following equation:

CE = 1/Q

where Q is the number of cells counted within a countingframe and the CE should be less than 0.10.

Creation of a Topographic MapConvert sample counts per graticule area to counts per

square millimetre (fig. 7d). Examine the calibrated cellcounts and look for patterns in the cell distribution. Drawiso-density lines or contours that connect areas with simi-lar cell densities. If an iso-density line indicates a valuebetween two counts, draw the line to fall between the two

points. The accurate position of this point can be locatedby extrapolation using the lines on the graph paper. Theseparation of the iso-density contours is arbitrary but mustreflect changes in density across the whole retina (fig. 7e).

Locate areas with higher cell densities such as an areacentralis, fovea or horizontal/visual streak. Recount areasof high cell density with a smaller distance between sam-pling areas to determine the rate of change in density.

Find the peak cell density (in all specializations if morethan one) to be used in subsequent calculations of peakspatial resolving power. Higher cell counts may be foundalong radial cuts. The combination of the downwardpressure that occurs as a result of the radial cuts and thereduced thickness of the peripheral retina where shrink-

age may be greater results in cell densities being artifi-cially elevated. Therefore, make sure to use a new scalpelblade or very sharp scissors for making peripheral slitsand avoid counting along radial cuts.

Draw all iso-density contours as smoothed lines eitherby hand or preferably using a graphics program such asAdobe Illustrator (Adobe Systems Inc., USA) or the opensource software Inkscape. The final topography mapshould also contain the retinal outline, the position ofnatural landmarks (e.g. optic nerve head, falciform pro-cess, pecten, conus), the retinal orientation and a scale bar(fig. 7f).

Calculation of Spatial Resolving PowerFor teleosts, use Matthiessenss ratio [Matthiessen,

1886]. The distance from the centre of a lens to the retina(PND) is 2.55 times the radius (r) of the lens, as follows:

PND = 2.55 *r

For birds, the PND is approximately 0.60 times theaxial length (L) of the eye [Hughes, 1977; Martin, 1993]:

PND = 0.60 *L

The angle () subtending 1 mm on the retina is equal

to

tan = 1 mm/PND

Spatial resolution is then calculated by obtaining thenumber of cells subtended by 1 degree of visual arc:

cells per degree = density at area of peak cell distribution/PND

At least 2 cells are needed to distinguish the dark andlight boundaries that make up 1 cycle of grating of thehighest resolvable frequency:

cycles per degree = cells per degree/2

For more detailed information on calculating spatialresolving power, see the studies of Collin and Pettigrew[1989] and Pettigrew et al. [1988].

Sample ExperimentThe retinal wholemount technique is an invaluable

method to visualize the retina of an animal. By examin-


15/19


ing the retina in toto, numerous features, including thetopographic arrangement and density of neurons, thepeak spectral sensitivities of photoreceptors and the loca-tion, size and shape of fundal landmarks, can be ascer-tained. These data can then be used to elucidate the neu-roethology of the animal as demonstrated by the follow-ing archerfish example.

The rather unusual foraging behaviour of the archer-fish (T. chatareus)is mirrored in its retinal topography. T.chatareuscaptures its prey by spitting out a stream of wa-ter to knock insects into the water, where they can be cap-tured. The different refractive indices of water and airmake this prey capture technique optically challenging,as the archerfish must compensate for the bending oflight during both the prey attack and prey recovery phas-

es [Temple, 2007]. We analysed the retinal topography ofthe archerfish by closely following the retinal whole-mount technique and found 3 retinal specializations thatproject upwards and forwards for viewing targets bothabove and below the water. Briefly, archerfish were dark-adapted for 2 h, then the eyes were removed and fixed,and finally the retina was extracted. Due to the presence

of areas of high cell density where the RPE was difficultto remove, the retinas were bleached as described above.

We wholemounted retinas for photoreceptor visualiza-tion and measurements of changes in density. Three reti-nal specializations were identified, 2 of low (fig. 8a) and 1of high (fig. 8b) relative density. Next, we developed topo-graphic maps of cone photoreceptor distributions and de-termined that the ventral retina had higher photoreceptor

a b

c

d e

f

C

olorversiona

vailable

online

g

Fig. 8.Sample results from the retina of an archerfish, T. chatareus.aLow-density pho-toreceptor distribution. The arrowhead indicates a double cone, while the asterisk indi-cates a single cone; the surrounding cells are rods. bHigh-density photoreceptor distri-bution. c Topographic map of combined double-cone and single-cone distributions.dLow-density ganglion cell (GC) distribution. eHigh-density ganglion cell distribution.fTopographic map of ganglion cell distribution.

gPoorly mounted retina with photore-ceptors lying on their sides. Note that the inner segment (IS) and outer segment (OS) are

visible in roughly the same plane.


16/19


Brain Behav Evol16

densities (greater than 22,500 cells/mm2; fig. 8c). The ven-tral area, which is aligned with the projection of Snellswindow, contains 3 smaller areas of even higher cell den-sities (each greater than 40,000 cells/mm2). Temporal andnasal acute zones that allow the archerfish to look out forpredators or prey in the rostral and caudal regions of their

visual field, respectively, were discovered in addition to aventral area (greater than 50,000 cells/mm2) that alignswith the spitting angle and is used to view aerial targets.By flipping and then staining the retina, we were able tovisualize the ganglion cell distribution (fig. 8d, e) in thesame eye. We found the ganglion cell distribution to besimilar to that of the photoreceptors, with the highestdensities occurring in the ventral region of the retina(fig. 8f). At this location, convergence ratios betweencones and ganglion cells were found to be nearly 1: 1, con-firming the functional significance of this region in sub-serving high visual acuity in the behaviour of the archer-

fish, which preys on small insects above the water andshoots them down with a small stream of water.

Discussion

The accessibility of the vertebrate retina was first rec-ognized over 120 years ago [Chievitz, 1889; Dogiel, 1891;Dogiel, 1895], when the retina was preserved and extract-ed from the eyecup in its entirety and used primarily fortopographic analysis of neurons. By carefully freeing theretina from the underlying RPE and choroidal tissue, the

retinal tissue could be wholemounted flat onto a glassslide to visualize cell somata and their spatial organiza-tion. In the early years, the technique was particularlyuseful for staining individual neurons using the Golgitechnique [Golgi, 1885] or silver impregnation [Rushton,1959] but was later modified, whereby, after an infusionof methylene blue, entire populations of retinal cellscould be visualized. This allowed for some of the first ex-aminations of retinal cell topography.

Selective pressures on visual function are reflected inthe diversity of specializations in the retina, which areareas devoted to high-acuity vision. The spacing of reti-

nal neurons is smallest within these retinal specializa-tions, yielding high sampling of an image (i.e. high-celldensity areas with high visual resolution). There are dif-ferent types of retinal specializations (e.g. areae, fovea,streaks) that have been identified in almost all vertebratesexamined. An area centralis is a concentric increase inganglion cell density towards the centre of the retina andis present in many species of mammals [Kolb and Wang,

1985; Murayama et al., 1995; Schmid et al., 1992]. A foveais characterized by a displacement of the inner layers ofthe retina and is associated with an increased packing ofphotoreceptors with elongated and smaller-diameter out-er segments beneath the foveal pit, as well as high densi-ties of ganglion cells in the perifoveal region. A fovea can

be convexiclivate, with steep sloping sides (found in somefishes and birds), or concaviclivate, with a shallow de-pression (found in monkeys and humans). Some preda-tory birds possess two foveae, namely a temporal fovea,which subtends the binocular visual space, and a mon-ocular fovea, which projects close to the laterally pointingoptical axis [Moroney and Pettigrew, 1987]. A horizontalstreak is an area of high ganglion cell density locatedacross the central region of the retina and is common inspecies that occupy habitats where the visual field is pre-dominated by a horizon (the air-land or air-water inter-face for terrestrial species or the water-substrate interface

for aquatic species), such as some coral reef fish, ostrichesand hyenas, among others.

When one dissects an eye from a previously unstudiedorganism, there are a number of gross morphological dif-ferences to be taken into account. These include the fol-lowing: (1) the position and shape of the optic nerve head,including multiple [Dunn-Meynell and Sharma, 1987,1988] and elongated [Collin et al., 2000] optic nerveheads; (2) any remaining scars due to the healing (or par-tial healing) of the embryonic fissure or falciform process[Collin and Pettigrew, 1988a]; (3) the shape and depth ofany foveal pit or depression in the retina [Collin and Col-

lin, 1999; Collin et al., 2000; Moroney and Pettigrew,1987; Wood, 1917]; (4) retinal thickening(s) across the ret-inal meridian typically indicating the position of one ormore horizontal/visual streaks [Munk, 1970]); (5) differ-ences in the size, shape and attachment of the lens [Gus-tafsson et al., 2010; Khorramshahi et al., 2008]; (6) thepresence or absence of vitreal vascularization, a conus ora pecten [Collin, 1989a; Hanyu, 1962; Nguyen, 1970;Smith et al., 1996; Yu et al., 2009], and (7) colored eyeshine produced by any tapetal material underlying theretina to increase visual sensitivity [Braekevelt, 1986,1990; Collin and Collin, 1993; Nicol, 1981]. The presence

of these morphological and, in many cases, species- ortaxon-specific variables presents technical difficulties.We hope this review al leviates many of these issues by ourmodifications to Stones [1981] techniques in terms of fix-ation, perfusion, removal of the vitreous humour andstaining of the retinal cells with cresyl violet.

The viscosity of the vitreous humour appears to be par-ticularly different across taxa, which may be due to the


17/19


18/19


Brain Behav Evol18

nia sp. that possesses 28 banks of rods underlying a deepconvexiclivate fovea [Locket, 1985]. This approach yieldslow-resolution topographicinformation but high-resolu-tion information regarding the arrangement, packingand density of neurons.

By making available the details of an enhanced whole-

mount technique based on a wider range of species, wehope to continue to promote the use of this technique.The wholemount technique is a powerful tool in com-parative neurobiology and visual ecology. New topo-graphic maps can be submitted to the Retinal Topogra-phy Database (http://www.retinalmaps.com.au/) for fu-ture comparative analyses on the evolution of the ver-tebrate visual system.

Acknowledgments

We would like to thank Tom Gallagher, Jeff Lucas, MeganGall, Lauren Brierley, Patrice Baumhardt, Jacquelyn Randolet andNathan Robinson for providing useful comments on an earlierversion of the draf t. We would also like to thank Joo Paulo Co-imbra for his useful discussion and instructions for stereology.

E.F.-J. and B.A.M. were partially supported by the National Sci-ence Foundation (IOS-0641550/0937187 to E.F.-J.). S.P.C. wouldlike to thank the Australian Research Council and the WesternAustralia State Government for their continued support. S.E.T.was supported by postdoctoral fellowships from The Universityof Queensland and the Natural Sciences and Engineering Re-search Council of Canada. J.F.P.U. was supported by an Endeav-our International Postgraduate Research Scholarship and a Uni-versity of Queensland International Living Allowance Scholar-ship.

References

Bailes HJ, Trezise AEO, Collin SP (2006): Thenumber, morphology, and distribution ofretinal ganglion cells and optic axons in theAustralian lungfish Neoceratodus forsteri(Kreff t 1870). Vis Neurosci 23: 257273.

Bowmaker JK, Heath LA, Wilkie SE, Hunt DM(1997): Visual pigments and oil dropletsfrom six classes of photoreceptor in the reti-nas of birds. Vision Res 37: 21832194.

Brach V (1977): The functional significance ofthe avian pecten: a review. The Condor 79:321327.

Braekevelt CR (1986): Fine structure of the tape-tum cellulosum of the grey seal (Halichoerusgrypus).Acta Anat (Basel) 127: 8187.

Braekevelt CR (1990): Fine structure of the felinetapetum lucidum. Anat Histol Embryol 19:

97105.Chelvanayagam DK (2000): Interpreting the dis-

tortion associated with a retinal whole-mount. J Theor Biol 205: 443455.

Chievitz JH (1889): Untersuchungen ber dieArea centralis retinae. Arch Anat Physiol139196.

Coggeshall RE, Lekan HA (1996): Methods fordetermining numbers of cells and synapses:a case for more uniform standards of review.J Comp Neurol 364: 615.

Collin SP (1989a): Topographic organization ofthe ganglion cell layer and intraocular vas-cularization in the retinae of two reef tele-osts. Vision Res 29: 765775.

Collin SP (1989b): Topography and mor phologyof retinal ganglion cells in the coral trout

Plectropoma leopardus(Serranidae): a retro-grade cobaltous-lysine study. J Comp Neurol281: 143158.

Collin SP, Collin HB (1993): The visual system ofthe Florida garfish, Lepisosteus platyrhincus(Ginglymodi). I. Retina. Brain Behav Evol42: 7797.

Collin SP, Collin HB (1999): The foveal photore-ceptor mosaic in the pipefish, Corythoich-thyes paxtoni(Syngnathidae, Teleostei). His-tol Histopathol 14: 369382.

Collin SP, Davies WL, Hart NS, Hunt DM (2009):The evolution of early vertebrate photore-ceptors. Philos Trans R Soc Lond B Biol Sci364: 29252940.

Collin SP, Hart NS, Wallace KM, Shand J, PotterIC (2004): Vision in the southern hemispherelampreyMordacia mordax: spatial distribu-tion, spectral absorption characteristics, andoptical sensitivity of a single class of retinalphotoreceptor. Vis Neurosci 21: 765773.

Collin SP, Lloyd DJ, Wagner H-J (2000): Foveatevision in deep-sea teleosts: a comparison ofprimary visual and olfactory inputs. PhilosTrans R Soc Lond B Biol Sci 355: 13151320.

Collin SP, Northcutt RG (1995): The visual sys-tem of the Florida garfish, Lepisosteus platy-rhincus (Ginglymodi). IV. Bilateral projec-

tions and the binocular visual field. BrainBehav Evol 45: 3443.

Collin SP, Pettigrew JD (1988a): Retinal topogra-phy in reef teleosts. II. Some species withprominent horizontal streaks and high-den-sity areae. Brain Behav Evol 31: 283295.

Collin SP, Pettigrew JD (1988b): Retinal ganglioncell topography in teleosts: a comparison be-tween Nissl-stained material and retrogradelabelling from the optic nerve. J Comp Neu-rol 276: 412422.

Collin SP, Pettigrew JD (1989): Quantitativecomparison of the limits on visual spatialresolution set by the ganglion cell layer intwelve species of reef teleosts. Brain BehavEvol 34: 184192.

Collin SP, Trezise AEO (2004): The origins of

colour vision in vertebrates. Clin Exp Op-tom 87: 217223.

Collin SP, Trezise AE (2006): Evolution of colourdiscrimination and its implications for visualcommunication; in Ladich F, Collin SP, MollerP, Kapoor BG (eds): Communication in Fish-es. Enfield, Science Publishers, pp 303335.

Dacey DM, Peterson MR (1992): Dendritic fieldsize and morphology of midget and parasolganglion cells of the human retina. Proc NatlAcad Sci USA 89: 96669670.

Dieterich CE, Dieterich HJ, Hildebrand R (1976):Comparative electron microscopic studies onthe conus papillaris and its relationship to theretina in night a nd day active geckos. GraefesArch Clin Exp O phthalmol 200: 279292.

Dogiel AG (1891): Ueber die nervsen Elementein der Retina des Menschen. Arch Mikr Anat38: 317344.

Dogiel AG (1895): Die Retina der Vogel. ArchMikr Anat 44: 622648.

Dolan T, Fernndez-Juricic E (2010): Retinalganglion cell topography of five species ofground-foraging birds. Brain Behav Evol 75:111121.

Dunlop S, Moore S, Beazley L (1997): Changingpatterns of vasculature in the developingamphibian ret ina. J Exp Biol 200: 24792492.

Dunn-Meynell AA, Sharma SC (1987): Visualsystem of the channel catfish (Ictaluruspunctatus). II. The morphology associatedwith the multiple optic papillae and retinalganglion cell distribution. J Comp Neurol257: 166175.

Dunn-Meynell AA, Sharma SC (1988): Visualsystem of the channel catfish (Ictaluruspunctatus).III. Fiber order in the optic nerveand optic tract. J Comp Neurol 268: 299312.

Ehrlich D (1981): Regional specialization of thechick retina as revealed by the size and den-sity of neurons in the ganglion cell layer. JComp Neurol 195: 643657.

Fernndez-Juricic E, Gall MD, Dolan T,ORourke C, Thomas S, Lynch JR (2011): Vi-sual systems and vigilance behaviour of two

ground-foraging avian prey species: white-crowned sparrows and California towhees.Anim Behav 81: 705713.

Fernndez-Juricic E, Gall MD, Dolan T, TisdaleV, Martin GR (2008): The visual fields of twoground-foraging birds, House Finches andHouse Sparrows, al low for simultaneous for-aging and anti-predator vigilance. Ibis 150:779787.


19/19


Fileta JB, Huang W, Kwon GP, FilippopoulosT, Ben Y, Dobberfuhl A, Grosskreutz CL(2008): Efficient estimation of retinal gan-glion cell number: a stereological approach.J Neurosci Methods 170: 18.

Frank BL, Goldberg S (1983): Multiple optic fiberpatterns in the catfish retina. Invest Oph-thalmol Vis Sci 24: 14291432.

Garamszegi LZ, Moller AP, Erritzoe J (2002):Coevolving avian eye size and brain size inrelation to prey capture and nocturnality.Proc Biol Sci 269: 961967.

Golgi C (1885): Sulla fina anatomia degli organicentrali del sistema nervoso. Reggio Emilia,Stefano Calderini e Figlio.

Gustafsson OS, Ekstrm P, Krger RH (2010): Afibrous membrane suspends the multifocallens in the eyes of lampreys and Africanlungfishes. J Morphol 271:980989.

Hanyu I (1962): Intraocular vascularization insome f ishes. Can J Zool 40: 87106.

Hart NS (2001): Variations in cone photorecep-tor abundance and the visual ecology ofbirds. J Comp Physiol A 187: 685697.

Hart NS (2002): Vision in the peafowl (Aves:

Pavo cristatus). J Exp Biol 205: 39253935.Hart NS, Partridge JC, Cuthill IC (1999): Visualpigments, cone oil droplets, ocular mediaand predicted spectral sensitivity in the do-mestic turkey (Meleagris gallopavo).VisionRes 39: 33213328.

Hayes BP, Holden AL (1983): The distribution ofdisplaced ganglion-cells in the retina of thepigeon. Exp Brain Res 49: 181188.

Hughes A (1977): The topography of vision inmammals of contrasting life style: compara-tive optics and retinal organisation; inCrescitelli F (ed): The Visual System of Ver-tebrates. Berlin, Springer, vol 2, pp 613756.

Khorramshahi O, Schartau JM, Krger RHH(2008): A complex system of ligaments and amuscle keep the crystalline lens in place in

the eyes of bony fishes (teleosts). Vision Res48: 15031508.

Koch PC, Seebacher C, Hess M (2010): 3D-to-pography of cell nuclei in a vertebrate retina a confocal and two-photon microscopicstudy. J Neurosci Methods 188: 127140.

Kolb H, Wang HH (1985): The distributionof photoreceptors, dopaminergic amacrinecells and ganglion cells in the retina of theNorth American opossum (Didelphis virgi-niana).Vision Res 25:12071221.

Levine JS, Macnichol EF, Collins BA, Kraft T(1979): Intraretinal distribution of cone pig-ments in certain teleost fishes. Science 204:523526.

Lichstein HC, Soule MH (1944): Studies of theeffect of sodium azide on microbic growth

and respiration. J Bacteriol 47: 221230.Litherland L, Collin SP (2008): Comparative vi-

sual function in elasmobranchs: spatial ar-rangement and ecological correlates of pho-toreceptor and ganglion cell distributions.Vis Neurosci 25: 549561.

Locket NA (1985): The multiple bank rod foveaof Bajacalifornia drakei, an Alepocephaliddeep-sea teleost. Proc R Soc Lond B Biol Sci224: 722.

Martin GR (1984): The visual fields of the tawnyowl, Strix alucoL. Vision Res 24: 17391741.

Martin GR (1993): Producing the image; in Zei-gler HP (ed): Vision, Brain and Behaviour inBirds. Cambridge, MIT Press, pp 524.

Matthiessen L (1886): Ueber den physikalisch-optischen Bau des Auges der Cetaceen undder Fische. Pflgers Arch Ges Physiol 38:

521528.Moroney MK, Pettigrew JD (1987): Some obser-vations on the visual optics of kingf ishers(Aves, Coraciformes, Alcedinidae). J CompPhysiol A 160: 137149.

Munk O (1970): On the occurrence and signifi-cance of horizontal band-shaped retinal ar-eas in teleosts. Vidensk Meddr Dansk NaturhForen 133: 85120.

Murayama T, Somiya H, Aoki I, Ishii T (1995):Retinal ganglion-cell size and distributionpredict visual capabilities of Dalls porpoise.Mar Mammal Sci 11: 136149.

Nguyen J (1970): The conus papillaris of reptiles.I. Ultrastructure in oplurus Oplurus cyalurus(Iguanidae). Z Mikr Anat Forsch 82: 1728.

Nicol JAC (1981): Tapeta lucida of vertebrates; in

Eroch JH, Tobey FL Jr (eds): Vertebrate Pho-toreceptor Optics. New York, Springer, pp401431.

Ollivier FJ, Samuelson DA, Brooks DE, LewisPA, Kallberg ME, Komaromy AM (2004):Comparative mophology of the tapetum lu-cidum (among selected species). Vet Oph-thamol 7: 1122.

ORourke CT, Hall MI, Pitlik T, Fernndez-Ju-ricic E (2010): Hawk eyes. I. Diurnal raptorsdiffer in visual fields and degree of eye move-ment. PLoS One 5:e12802.

Pettigrew JD, Dreher B, Hopkins CS, McCall MJ,Brown M (1988): Peak density and distribu-tion of ganglion cells in the retinae of micro-chiropteran bats: implications for visual acu-ity. Brain Behav Evol 32: 3956.

Rushton WAH (1959): Giant ganglion cells in thecats retina. J Physiol 111: 2627P.

Schiviz AN, Ruf T, Kuebber-Heiss A, Schubert C,Ahnelt PK (2008): Retinal cone topographyof artiodactyl mammals: influence of bodyheight and habitat . J Comp Neurol 507: 13361350.

Schmid KL, Schmid LM, Wildsoet CF, PettigrewJD (1992): Retinal topography in the koala(Phascolarctos cinereus). Brain Behav Evol39:816.

Segev R, Schneidman E, Goodhouse J, Berry MJ(2007): Role of eye movements in the retinalcode for a size discrimination task. J Neuro-physiol 98: 13801391.

Smith BJ, Smith SA, Braekevelt CR (1996): Finestructure of the pecten oculi of the barred owl

(Strix varia).Histol Histopathol 11: 8996.Somiya H (1980): Fishes with eye shine: func-

tional morphology of guanine type tapetumlucidum. Mar Ecol Prog Ser 2: 926.

Stone J (1981): The Wholemount Handbook. AGuide to the Preparation and Analysis ofRetinal Wholemounts. Sydney, MaitlandPublications.

Takechi M, Kawamura S (2005): Temporal andspatial changes in the expression pattern ofmultiple red and green subtype opsin genesduring zebrash development. J Exp Biol208: 13371345.

Takei S, Somiya H (2002): Guanine-type retinaltapetum and ganglion cell topography in theretina of a carangid fish, Kaiwarinus equula.

Proc Biol Sci 269: 7582.Tandrup T (1993): A method for unbiased andefficient estimation of number and mean

volume of specified neuron subtypes in ratdorsal-root ganglion. J Comp Neurol 329:269276.

Temple SE (2007): Effect of salinity on the refrac-tive index of water: considerations for archerfish aerial vision. J Fish Biol 70: 16261629.

Temple SE (2011): Why different regions of theretina have different spectral sensitivities: areview of mechanisms and functional signif-icance of intraretinal variability in spectralsensitivity in vertebrates. Vis Neurosci 28:281293.

Temple S, Hart NS, Marshall NJ, Collin SP(2010): A spitting image: specializations in

archerfish eyes for vision at the interface be-tween a ir and water. Proc Biol Sci 277: 26072615.

Thanos S, Pavlidis C, Mey J, Thiel HJ (1992): Spe-cific transcellular staining of microglia inthe adult rat after traumatic degeneration ofcarbocyanine-filled retinal ganglion cells.Exp Eye Res 55: 101117.

Theiss S, Lisney T, Collin S, Hart N (2007): Col-our vision and visual ecology of the blue-spotted maskray, Dasyatis kuhlii Mller &Henle, 1814. J Comp Physiol A NeuroetholSens Neural Behav Physiol 193: 6779.

Vaney DI, Collin SP, Young HM (1989): Dendrit-ic relationships between cholinergic ama-crine cells and direction-selective retinalganglion cells; in Weiler R, Osbourne NN

(eds): The Neurobiology of the Inner Retina.NATO ASI Series/Cell Biology. Berlin,Springer, pp 157168.

Vidal-Sanz M, Villegas-Prez MP, Bray GM,Aguayo AJ (1988): Persistent retrograde la-beling of adult rat retinal ganglion cells withthe carbocyanine dye DiI. Exp Neurol 102:92101.

Wilhelm M, Straznicky C (1992): The topo-graphic organization of the retinal ganglioncell layer of the lizard Ctenophorus nuchalis.Arch Histol Cytol 55: 251259.

Wong KW, Adolph AR, Dowling JE (2005): Ret-inal bipolar cell input mechanisms in giantdanio. I. Electroretinographic analysis. JNeurophysiol 93: 8493.

Wong ROL, Meister M, Shatz CJ (1993): Tran-

sient period of correlated bursting activityduring development of the mammalian reti-na. Neuron 11: 923938.

Wood CA (1917): The Fundus Oculi of Birds, Es-pecially as Viewed by the Ophthalmoscope.A Study in Comparative Anatomy and Phys-iology. Chicago, Lakeside Press.

Yu CQ, Schwab IR, Dubielzig RR (2009): Feed-ing the vertebrate retina from the Cambri-an to the Tertiary. J Zool 278: 259269.

Montagem de Retina

Documents

Transcript of Montagem de Retina