R E S EA R C H A R T I C L E

Photoreceptor Topography and Spectral Sensitivity in the Common Brushtail Possum (Trichosurus vulpecula) Lisa M. Vlahos,1,2* Ben Knott,3 Krisztina Valter,1,4 and Jan M. Hemmi1,2,5 1

ARC Centre of Excellence in Vision Science, Australian National University, Canberra, ACT 0200, Australia Research School of Biology, College of Medicine, Biology and Environment, Australian National University, Canberra, ACT 0200, Australia 3 Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University, Geelong, VIC, 3217, Australia 4 Medical School and the John Curtin School of Medical Research, College of Medicine, Biology and Environment, Australian National University, Canberra, ACT 0200, Australia 5 School of Animal Biology and the UWA Oceans Institute, University of Western Australia, Crawley, WA 6009, Australia 2

ABSTRACT Marsupials are believed to be the only non-primate mammals with both trichromatic and dichromatic color vision. The diversity of color vision systems present in marsupials remains mostly unexplored. Marsupials occupy a diverse range of habitats, which may have led to considerable variation in the presence, density, distribution, and spectral sensitivity of retinal photoreceptors. In this study we analyzed the distribution of photoreceptors in the common brushtail possum (Trichosurus vulpecula). Immunohistochemistry in wholemounts revealed three cone subpopulations recognized within two spectrally distinct cone classes. Longwavelength sensitive (LWS) single cones were the largest cone subgroup (67–86%), and formed a weak horizontal visual streak (peak density 2,106 6 435/mm2) across the central retina. LWS double cones were strongly concentrated ventrally (569 6 66/mm2), and created a "negative" visual streak (134 6 45/mm2) in INDEXING TERMS: marsupial; microspectrophotometry

mammal;

color

Research over the past 15 years has greatly expanded our understanding of color vision in marsupials: the first non-primate mammals known with up to three spectrally distinct cone classes. Eutherian mammals typically have a duplex retina with rods for high sensitivity and low acuity vision during scotopic conditions, and two cone classes for low sensitivity and high acuity vision during photopic conditions (Ebrey and Koutalos, 2001). These two cone classes are typically longwavelength sensitive (LWS) and short-wavelengthsensitive (SWS; Peichl, 2005). The ability to compare C 2014 Wiley Periodicals, Inc. V

the central retina. The strong regionalization between LWS cone topographies suggests differing visual functions. Short-wavelength sensitive (SWS) cones were present in much lower densities (3–10%), mostly located ventrally (179 6 101/mm2). A minority population of cones (0–2.4%) remained unlabeled by both SWS- and LWS-specific antibodies, and may represent another cone population. Microspectrophotometry of LWS cone and rod visual pigments shows peak spectral sensitivities at 544 nm and 500 nm, respectively. Cone to ganglion cell convergences remain low and constant across the retina, thereby maintaining good visual acuity, but poor contrast sensitivity during photopic vision. Given that brushtail possums are so strongly nocturnal, we hypothesize that their acuity is set by the scotopic visual system, and have minimized the number of cones necessary to serve the ganglion cells for photopic vision. J. Comp. Neurol. 522:3423–3436, 2014. C 2014 Wiley Periodicals, Inc. V

vision;

cone;

rod;

retina;

immunohistochemistry;

Grant sponsor: Australia Research Council Future Fellowship (to J.M.H.); Grant sponsor: Australian Research Council Centre of Excellence in Vision Science. *CORRESPONDENCE TO: Lisa M. Vlahos, ARC Centre of Excellence in Vision Science, Division of Evolution, Ecology and Genetics, Research School of Biology, College of Medicine, Biology and Environment, Australian National University, Canberra, ACT 0200, Australia. E-mail: [email protected] Received November 2, 2013; Revised April 9, 2014; Accepted April 9, 2014. DOI 10.1002/cne.23610 Published online April 16, 2014 in Wiley (wileyonlinelibrary.com)

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The Journal of Comparative Neurology | Research in Systems Neuroscience 522:3423–3436 (2014)

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the output signals of the two cone classes provide most mammals with dichromatic color perception (Jacobs, 1981). Recent evidence suggests that some Australian marsupial species may have three cone types, serving as the basis for trichromatic color vision. Immunohistochemistry and microspectrophotometry (MSP) in four marsupial species, the fat-tailed dunnart (Sminthopsis crassicaudata), honey possum (Tarsipes rostratus), quokka (Setonix brachyurus), and quenda (Isoodon obesulus) found a third, middle-wavelength sensitive (MWS) cone type (Arrese, 2002; Arrese et al., 2002b, 2003, 2005; Ebeling et al., 2010). The presence and function of these MWS cones remains debatable, due to strong similarities between the spectral sensitivities of MWS cones and rod visual pigments for all tested species, and the lack of immunolabeling with any specific antibody (Ebeling et al., 2010). In contrast, the Australian tammar wallaby, Macropus eugenii (Hemmi, 1999; Hemmi and Gr€unert, 1999; Deeb et al., 2003; Ebeling et al., 2010; Ebeling and Hemmi, 2014), and several of the American opossums (Ahnelt et al., 1995; Hunt et al., 2003; Jacobs and Williams, 2010; Palacios et al., 2010; Gutierrez et al., 2011) have been shown to possess only two spectrally distinct cone classes, resulting in dichromatic color vision. Interestingly, there is no genetic evidence for a third cone type in any marsupials. Only the SWS and LWS cone opsin genes have been found in the marsupials using molecular genetic techniques (Deeb et al., 2003; Strachan et al., 2004; Cowing et al., 2008; Hunt et al., 2009b). Marsupials occupy a diverse range of habitats, so it is likely that there is considerable variation in the presence, density, and distribution of photoreceptor classes. Currently, it is still uncertain whether or not the tammar wallaby is the only Australian marsupial with dichromacy (Hemmi and Gr€unert, 1999; Ebeling and Hemmi, 2014), or if it is represented in a number of Australian species. In addition, the retinal topography of all classes and morphological types of cones is still unknown in most Australian species. The topographical distribution of the third spectral cone class has not been described for any of the trichromatic marsupials. Analyzing both the density and distribution of cones not labeled with SWS and LWS-specific antisera across the retina may provide more insight into the significance of this cone population. While double cones are absent in placental mammals, both single and double cone types are common in monotremes and marsupials (O’Day, 1938; Young and Pettigrew, 1991; Ahnelt et al., 1995; Hemmi and Gr€unert, 1999; Arrese et al., 2003). Since double and single cones contain the same LWS visual pigment in marsupials, previous studies typically counted both cell types to generate maps of the total

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population of LWS cones. Some studies, however, described differences in the density of single and double cones across the retina. The tammar wallaby and South American marsupial Thylamys elegans were noted to have more double cones ventrally, with a small reduction in double cones centrally (Hemmi and Gr€unert, 1999; Palacios et al., 2010). Arrese et al. (2003) also found more double than single cones in the mid to far peripheral regions of the honey possum and fat-tailed dunnart retina. Double cones have only been analyzed topographically in the South American opossum Didelphis marsupialis (Ahnelt et al., 1995). In this species, the double cones were concentrated in the ventral retina, with a notable decrease in double cones in the same region as the single cones visual streak. Currently, the function of double cones is the subject of debate: there is both evidence for (Neumeyer, 1986; Hughes et al., 1998; Pignatelli et al., 2010) and against (Boehlert, 1978; Vorobyev and Osorio, 1998; Osorio et al., 1999) double cones being involved in color vision. Proposed alternative functions include motion, polarized light, or luminance detection (Young and Martin, 1984; Cameron and Pugh, 1991; Novales Flamarique et al., 1998; Vorobyev and Osorio, 1998). Further studies are clearly needed to expand our knowledge of Australian marsupial color vision, and ultimately provide a comprehensive description of photoreceptor topography and diversity across marsupials. In this study we investigate the topographic distribution, antibody specificity, and spectral characteristics of cones in the retina of the Australian common brushtail possum (Trichosurus vulpecula). This semi-arboreal marsupial is known to be exclusively nocturnal, and therefore may be a good candidate for a dichromatic color vision system. Deeb (2010) reported unpublished data, which determined that the common brushtail possum has two cone visual pigments, with maximum absorbance wavelengths (kmax) of 360 nm and 556 nm, respectively. It is unclear, however, whether both visual pigments were determined by microspectrophotometry, amino-acid sequencing, or both. The kmax of the rod visual pigment has not been established. The brushtail possum’s ganglion cell distribution has previously been documented to form a weak visual streak centrally (Freeman and Tancred, 1978). The topographic distribution of cone subtypes can often be different from that of ganglion cells, and this variation can reflect the sensitivity or acuity pressures associated with regional specializations in a species’ visual environment (Ahnelt et al., 1995; Hemmi and Gr€unert, 1999). In this study we provide a comprehensive description of all cone distributions, using two immunolabeling methods to ensure consistent results. We also discuss how these results may be related to the brushtail possum’s visual ecology.

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TABLE 1. Primary Antibodies Antibody

Host

LWS opsin

Rabbit polyclonal

SWS opsin

Rabbit polyclonal

Rhodopsin

Mouse monoclonal

Peanut agglutinin (PNA)

Lectin peanut agglutinin from Arachis hypogaea

Immunogen Raised against the last 38 amino acids of the C-terminus of the recombinant human red/green opsin (RQFRNCILQLFGKKVDDGSELSSASKTEVSSVSSVSPA). Raised against the last 42 amino acids of the recombinant human blue opsin (NKQFQACIMKM VCGKAMTDESDTCSSQKTEVSTVSSTQVGPN). Raised against last 9 amino acids of the C-terminus of bovine rhodopsin (TETSQVAPA). No immunogen; Binds to galactosyl (b-1,3) N-acetylgalactosamine.

MATERIALS AND METHODS Animals Four adult brushtail possums (two females and two males) were captured on the campus of the Australian National University (ANU). Animals were anesthetized with isoflurane (4–5% inhaled with oxygen at 1 L/min) before being euthanized with an intracardiac injection of sodium pentobarbitone (300 mg/ml, 1 ml/kg body weight) mixed with lignocaine (0.4–0.8 mg/5kg body weight). All experiments were approved by the ANU Animal Experimentation Ethics Committee under protocol A2011/004. Experiments also comply with the ACT Department of Territory and Municipal Services (License number LE2011320, LT2011489 and K8988) under the Nature Conservation Act (1980), and the VIC Department of Sustainability and Environment (Permit number 10005915 and 14047128) under the Wildlife Act (1975).

Immunohistochemistry Five eyes were immersed in 4% paraformaldehyde in 0.1M phosphate-buffered saline (0.1M PBS) for a minimum of 12 hours, then transferred to 0.1M PBS. Retinas were dissected as wholemounts with the photoreceptor cell layer facing upwards. Wholemount orientation was easily determined by the heavily pigmented epithelium in the ventral region of the brushtail possum retina (Buttery et al., 1990). Photographs were taken to allow easy identification of sample areas and to maintain correct orientation. For basic retinal morphology, pieces of retina not used for immunohistochemistry were embedded in Medcast resin, sectioned vertically (i.e. perpendicular to the retinal layers) at 1lm thickness, and stained with Toluidine Blue. Micrographs were taken with a Zeiss Axioskop light microscope (Carl Zeiss Vision, Germany). Immunohistochemistry was performed on four retinal wholemounts for cone topography analysis. The retinal pigment epithelium was first bleached to improve the

Source Chemicon/Millipore,Catalog No. AB5405, RRID:AB_177456 JH455 antibody donated by U. Gr€unert (University of Sydney, Australia), RRID:AB_2313107 Chemicon/Millipore,Catalog No. Rho 1D4, RRID:AB_2313039 Vector Labs,Catalog No. B-1075, RRID:AB_2313597

visualization of photoreceptors during confocal microscopy (Hemmi and Gr€unert, 1999). Retinae were incubated for up to 4 hours at room temperature in a solution of 1.8% NaCl, 30% H2O2, 3 ml H2O, and three drops of NH3, then washed with 0.1M PBS (1 3 30 minutes). Free-floating wholemounts were immersed in 30% sucrose for 24 hours at 4 C for cryoprotection, then shock-frozen and thawed three times for improved antibody penetration. They were preincubated in 10% normal goat serum (NGS) in a T-PBS solution (0.01% Triton X-100 with 0.1M PBS, pH 7.4) for 4 hours at 37 C to limit nonspecific binding. Wholemounts were then treated with a primary cone antibody solution for 3–4 days at 4 C. Two of the wholemounts were labeled with either the LWS opsin antibody AB5405 (1:1,000 dilution; Chemicon/Millipore, Temecula, CA; RRID:AB_177456), or the SWS opsin antibody JH455 (1:25,000 dilution; donation from U. Gr€unert, University of Sydney, Australia; RRID:AB_2313107; see Table 1). The remaining two wholemounts were double-labeled for both SWS and LWS cones by incubating the tissue in a mixture of antisera JH455 and AB5405. Any cones that remain unlabeled in marsupial retinae are typically interpreted to be a third cone population (Arrese et al., 2003, 2005; Ebeling et al., 2010). All primary cone antibodies were visualized by incubating with rabbit IgG conjugated with Alexa Fluor 594 (1:200; Invitrogen, La Jolla, CA; RRID:AB_141359) for 2 days at 4 C. Retinal wholemounts were then treated with biotinylated peanut agglutinin (PNA; 1:500; Vector Labs, Burlingame, CA; RRID:AB_2313597) for 2 days at 4 C to mark all cone photoreceptors. PNA was visualized by incubating for 24 hours at 4 C with streptavidin Cy2 (1:200; Jackson ImmunoResearch, West Grove, PA; RRID:AB_2307356). The PNA labeling image was converted from fluorescent green to blue by displaying the green PNA fluorescence through the blue image channel. This allowed for easier visualization between the targeted cone opsin labeling and PNA during analysis.

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A fifth retina was semithin plastic sectioned to check if there were any differences in the labeling frequency of cone opsin types seen in either wholemount or section immunohistochemistry, and therefore avoid any potential inferences based on a single type of methodology. Sections were also used to calculate rod densities along a dorsal to ventral transect and checked for coexpression of opsin antibodies. Pieces of retina were sectioned horizontally to the retina into 1-mm2 pieces along a dorsal to ventral transect, then dehydrated in a sequence of ethanol and acetone baths before being embedded in Medcast (Ted Pella, Redding, CA) resin blocks. Consecutive 0.5 lm thick horizontal sections (i.e., parallel to the retinal layers) were cut with an ultramicrotome and mounted on alternate glass slides. The resin was removed by immersing slides in a 1:1 solution of 1M NaOH and 100% EtOH for 15 minutes. Slides were then rinsed with 100% ethanol (1 3 5 minutes) and distilled H2O (3 3 10 minutes). Sections were preincubated with 1% NGS in T-PBS for 1 hour at 37 C before incubating with the primary antibodies for 24 hours at 4 C. Consecutive sections on alternate slides were treated with either the AB5405 (1:1,000) or JH455 (1:10,000) antibody. All slides were also treated with the monoclonal mouse antibody Rho1D4 (1:500; Chemicon/Millipore; RRID:AB_2313039). Sections were treated with secondary antibodies Alexa Fluor 594 (1:1,000) and mouse IgG conjugated with Alexa Fluor 488 (1:1,000; Invitrogen; RRID:AB_141367) for 24 hours at 4 C. Rhodopsin labeling was again converted from fluorescent green to blue for easier visual analysis between targeted rod and cone labeling. All antibodies were stored in 0.1% sodium azide at 4 C, until diluted with 1% NGS in TPBS right before application. Tissue was washed in TPBS (3 3 30 minutes) between each antibody application, and light-protected during incubation, rinses, coverslipping (glycerol gelatin), and storage. Omission of the primary antibodies from the immunostaining protocols resulted in no labeling, showing the specificity of fluorescent secondary antibodies.

Antibody characterization Table 1 summarizes the antigen, host, and source of each primary antibody used in this study. The rabbit polyclonal antibody AB5405 (Chemicon/Millipore; RRID:AB_177456) was raised against the last 38 amino acids of the human red/green opsin C-terminus (Wang et al., 1992). The rabbit polyclonal antibody JH455 was raised against the last 42 amino acids of the human blue visual pigment (Wang et al., 1992). JH455 (1:25,000) was kindly provided by U. Gr€unert (University of Sydney, Australia), which was originally donated by J.

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Nathans (Johns Hopkins University School of Medicine, Baltimore, MD; RRID:AB_2313107). This antibody can also be purchased commercially (Chemicon/Millipore, Cat. No. AB5407; RRID:AB_177457). Both AB5405 and JH455 antibodies were initially produced by Wang et al. (1992). DNA segments encoding the last 38 amino acids of the human red/green opsin and the last 42 amino acids of the human blue opsin were separately inserted into the polylinker of the T7 gene 10 expression vector pGEMEX (Promega, Madison, WI). Each cone opsin-derived peptide was produced as a Cterminal extension of the T7 gene 10 protein. The fusion proteins were purified and then used to immunize rabbits. Antisera were tested by immunofluorescent labeling of transiently transfected tissue culture cells expressing recombinant human cone visual pigments (Merbs and Nathans, 1992). Each exhibited labeling only in tissue culture cells transfected with the corresponding cDNA clone, but not in the untransfected cells (Merbs and Nathans, 1992). The specificity of the AB5405 antibody was tested by the manufacturer with western blot analysis using mouse retinal extracts (Chemicon/Millipore). Each analysis revealed a band of 40 kDa molecular weight, which corresponds with the respective sizes of the opsin protein. The antiserum JH455 stains a major band from 37 kDa to 40 kDa molecular weight on western blots in rodent and dog retina (Bobu et al., 2006; Gaillard et al., 2009; Genini et al., 2010). The commercially available version of JH455 stains the 39 kDa form of the molecule on western blot (Chemicon/Millipore, Cat. No. AB5407). Both AB5405 and JH455 antisera label the outer segments of specific cone types in the retina of several mammalian species (Hemmi and Gr€unert, 1999; Arrese et al., 2003, 2005; MacNeil and Gaul, 2008; Raven et al., 2008; Xu and Tian, 2008; Ebeling et al., 2010; Zeiss et al., 2011). The mouse monoclonal antibody Rho1D4 was developed against bovine rhodopsin by immunizing mice with bovine rod outer segment disk membranes (Molday and MacKenzie, 1983; MacKenzie et al., 1984; RRID:AB_2313039). A peptide comprising the last nine amino acids of the C-terminus was used to map the binding sites of Rho1D4. The specificity of the Rho1D4 antibody was tested by the manufacturer with a western blot analysis, which recognized 36 kDa monomers, dimmers, and trimers depending on sample preparation (Chemicon/Millipore). Rho1D4 has been effective to specifically label the rod outer segments in the retina of several mammals (Hicks and Barnstable, 1987; Frederick et al., 2001; Ebeling et al., 2010; Ji et al., 2012). The biotinylated PNA is a 110 kDa plant lectin isolated from Arachis hypogea (Vector Labs;

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Retinal specializations in brushtail possum

RRID:AB_2313597). PNA binds to galactosyl (b-1,3) Nacetylgalactosamine structures present in the interphotoreceptor matrix, which sheaths cone outer and inner segments, and cone pedicles (Johnson et al., 1986). PNA binds to interphotoreceptor matrix domains around the outer segments of cones in mammals (Blanks and Johnson, 1984; Ahnelt and Kolb, 2000), although labeling can vary based on composition differences in the matrix sheath of cone types (Blanks and Johnson, 1984; Szel et al., 1993).

Analysis Immunolabeled wholemounts were imaged using a Zeiss LSM 5 Pascal confocal microscope (Carl Zeiss Vision, Germany). Each sampled location was scanned down the entire photoreceptor layer by confocal z-stacking. Cells in the three wholemounts labeled with AB5405, or both JH455 and AB5405 antibodies were counted in 225 3 225 lm sampling frames. The frames in the fourth wholemount, labeled with JH455, were increased to 450 3 450 lm. This ensured that the lower density of SWS cones was accurately sampled. Counting frames were initially sampled at uniform and systematic 2-mm intervals, which were randomly determined by the placement of the retina on the slide. Sampling rates were then increased to 1-mm or 0.5-mm intervals in locations where the cone densities changed rapidly. Custom-made MatLab (MathWorks, Natick, MA; RRID:nlx_153890)-based digitizing software (J.M.H.) was used as an image analysis system to mark and analyze the position of cones. The digitizing software could import and use individual z-stack images for analysis, and also generate a 2D image summated from the z-stacks to create an overall image. Outer segments were initially marked by using the 2D image, and then checked against the individual z-stack images for each color channel. This was to ensure that any other segments that had a weak fluorescent signal were not missed during the image summation. Photoreceptors which were in contact with the bottom or left frame borders were excluded from the analysis in order to prevent a bias against larger-sized cells. Single and double cones were marked separately in all wholemounts, along with cones labeled or not labeled by primary antisera. An unlabeled cone was defined as a cone where the opsin-specific JH455 and AB5405 antibodies did not label the outer segment, but the presence of an intact outer segment was confirmed by PNA labeling bound on the cone matrix sheath surrounding the outer segment. Topographic maps were then plotted as spline-smoothed isodensity contour plots, showing regular intervals between the lowest and highest measured cell density. Immunohistochemistry in the semithin plastic sections were imaged with a Zeiss Apotome fluorescence

light microscope (Carl Zeiss Vision, Germany). Images were exported to Adobe Illustrator (CS3, Adobe Systems, San Jose, CA; RRID:nlx_157287) for analysis. Several 50 3 50 lm counting frames were placed at regular intervals along the dorsal to ventral transect. Frame locations from consecutive sections were aligned by matching various landmarks in each section to create a digital stack. Individual cones were tracked down the consecutive counting frame sections, which had undergone alternating AB5405 and JH455 labeling to check for coexpression of these antibodies. Rod densities were determined from one image in each digital stack. The convergence ratios of photoreceptors to ganglion cells was obtained by comparing cone and rod densities along the dorsal to ventral transect to that of known ganglion cell distributions (Freeman and Tancred, 1978). Where the convergence ratio drops to one or below, meaning that there are fewer or equal number of cones as ganglion cells, the cone counts provide an upper estimate of visual acuity in the transect. The number and position of sampled rods and cones was calculated with the same digitizing software used for topography analysis. A thin-plate spline function was fitted to the data points to interpolate between the measured locations.

Microspectrophotometry One possum was dark-adapted overnight before being euthanized under dim red light. Both eyes were transported in 0.1M PBS to Deakin University (Geelong, Australia), and analyzed over 2 days. Under infrared illumination, small pieces of retinal tissue were mounted between two glass coverslips in a drop of 0.1M PBS containing 10% sucrose. Retinal samples were examined using a computer-controlled single beam wavelength-scanning MSP. A 2 lm2 measuring beam was aligned to a targeted area, and then sequentially illuminated with monochromatic light from 750 nm to 350 nm at 2 nm intervals and then again from 351 nm to 749 nm. Light absorbance of the measuring beam at each wavelength was automatically saved to file. A baseline scan was recorded in a tissue-free area adjacent to the retinal sample, and automatically subtracted from subsequent scans. A prebleached sample scan was made by aligning the measuring beam to pass through the outer segment of a photoreceptor. Photoreceptors were targeted along the edge of the retinal sample, where most photoreceptor outer segments were lying horizontally. Outer segments were then bleached with broad-spectrum white light for up to 6 minutes. The "post-bleach" scan was measured to create difference spectra and confirm that the targeted outer segment contained a photo-labile, and not a

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Figure 1. Brushtail possum retinal morphology and photoreceptors. A: Vertical thin (1 lm) section from dorsal retina, stained with Toluidine blue. RPE: retinal pigment epithelium, OS: outer segments, IS: inner segments, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL: ganglion cell layer. The brushtail possum has colorless oil droplets located between the inner and outer segment. B: Horizontal semithin (0.5 lm) section immunolabeled with AB5405 (red) and Rho1D4 (blue). There is a dense population of rod photoreceptors, and sparse single (arrow) and double (arrowhead) cones present. C: Horizontal semithin (0.5 lm) section immunolabeled with JH455 (red) and Rho1D4 (blue) demonstrates the low density and irregularly spaced cone mosaic of SWS cones. Scale bars 5 20 lm.

photo-stable pigment. Cells were sampled from several locations of both retinae from one animal.

Analysis Scans were exported from the MSP software and analyzed in MatLab. An 11-point running average was employed to remove high frequency noise, and wavelengths below 400 nm were removed to improve the signal-to-noise ratio. Similar to Hart et al. (1998), only individual pre-bleach absorbance spectra that satisfied the following selection criteria were used during the analysis: 1) The shape of measurements shows a steady decline in absorbance towards the longer wavelengths; 2) Scans were free from obvious distortions in the long wavelength limb (between 70% and 30% normalized absorbance), which is caused by photoproduct accumulation; and 3) Scans were confirmed as a visual pigment measurement by the loss of absorbance after bleaching the cell with white light. The scans that met these criteria were then fitted with a Stavenga retinal pigment (A1)based template curve (Stavenga et al., 1993), which was then used to find the mean wavelength of maximum absorbance (kmax) for each visual pigment.

RESULTS Retinal structure and antibody specificity The overall retinal structure is consistent with a typical mammalian pattern, with rods forming the majority

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of the photoreceptors (Fig. 1; Ahnelt and Kolb, 2000). The outer nuclear layer (ONL) is the thickest of the retinal layers, associated with a strong rod presence (Fig. 1A). Both rods and cones are long and slender, a common feature in nocturnal mammalian retinae. Figure 1A also shows colorless oil droplets present between the inner and outer segment of cone photoreceptors. The presence of oil droplets in cones is consistent with other Australian marsupials studied to date (Hemmi and Gr€unert, 1999; Arrese et al., 2003. 2005). Immunohistochemistry on horizontal sections highlight the high rod (blue) densities present in the brushtail possum retina, and lower cone density (red, Fig. 1B,C). Similar to the other marsupials studied to date, brushtail possums have both single (arrows) and double (arrowheads) cones present (Figs. 1B, 2A). In double cones, the outer segment of the accessory cone is of similar size to that of the primary cone. The antisera/lectin used during both immunohistochemistry methods clearly label photoreceptors (Figs. 1B,C, 2A). Labeling of both rods and cones is characterized by a very strong fluorescent signal, with very little background labeling. Cone antibody labeling is restricted to the outer segments. The antiserum AB5405 recognized all double cones and a subpopulation of single cones, suggesting that their visual pigment belongs to the same LWS photoreceptor class (Figs. 1B). The antiserum JH455 recognized a sparse and irregularly spaced population of SWS single cones

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Retinal specializations in brushtail possum

Figure 2. Micrographs of retinal wholemount from ventral retina immunolabeled with AB5405 and JH455 (red) and PNA (blue). A: Both PNA and opsin antibodies label single (arrow) and double (arrowhead) cones. A small population of cones remains unlabeled (example shown in insert, yellow arrow). The outer segment of unlabeled cones is confirmed to be intact by the surrounding PNA-labeled cone matrix sheath (B), but is not labeled with the SWS and LWS-specific antibodies (C). Scale bars 5 20 lm.

(Fig. 1C). PNA typically labels either the entire cone matrix sheath, or is confined to the outer segment region (Fig. 2A). Double cones have a stronger PNA signal than single cones. The retinal wholemounts that are double-labeled with both LWS and SWS-specific antisera show a small number of cones, which remain unlabeled by the antisera (Fig. 2A, yellow arrow). The outer segment of these cones is not recognized by the LWS and SWS-specific antibodies, but the presence of an intact outer segment is inferred by the PNA labeling, which binds on the cone matrix sheath surrounding the outer segment (Fig. 2B,C). Unlabeled cones appear in both double-labeled retinal wholemounts, and in semithin plastic section immunohistochemistry. The authors did not observe any regular spacing of unlabeled cones. Coexpression of opsin antibodies has not been observed for any photoreceptors.

Photoreceptor topography and distribution Immunohistochemistry in retinal wholemounts revealed three cone subtypes recognized within two antibody-specific cone classes, and a small population of cones which were not recognized with any of the opsin antisera. Figure 3 shows representative wholemounts illustrating the topographic distribution of cone types in the retina. Figure 4 shows the distribution of photoreceptors along a dorsal to ventral transect, and thin-plate spline functions used to interpolate between data points. Single and double LWS cones show two distinct topographical distributions (Fig. 3A,B). The dis-

tribution of LWS single cones reveals a weak visual streak in the central retina, with two broad peaks in density located in both the temporal and nasal area (Fig. 3A). The streak itself extends more into the temporal region, suggesting that the nasal peak may be a variation of the streak rather than the main peak of the visual streak. The highest concentration of cells within the visual streak is on average 2,106 6 435 cones/ mm2 (n 5 2 retinae). A comparison of cell density across cone subtypes shows that, on average, LWS single cones represent 67–86% (n 5 3) of the total cone population (Fig. 4A). Meanwhile, LWS double cones form a "negative" visual streak in the same region as the LWS single cone visual streak, creating a loose step function from the dorsal to ventral retina (n 5 2, Fig. 3B). This "negative" visual streak has densities as low as the far periphery, at 134 6 45 cones/mm2, or 9% of the total cone population (n 5 3, Fig. 4A). The highest concentration of LWS double cones is 2–3 mm ventraltemporal from the optic disc, with an average of 569 6 66 cones/mm2. In the ventral retina, LWS double cones reach to almost 30% of the total cone population, and overall represent 17% of all cones in the retina (n 5 3, Fig. 4A). SWS cones are present in much smaller numbers than LWS cones, and only of single cone morphology (Fig. 3C). SWS cone topography show a slight increase in cell density from dorsal-nasal to ventral-temporal, reaching a maximum density of 179 6 101 cones/mm2 (n 5 2 retinae). Dorsal and central regions have a lower density of 64 6 52 cones/mm2 (n 5 2). Comparisons between cone photoreceptor types along the dorsal to ventral transect show that SWS cones reach 3–10% (n 5 3) of the total population (Fig. 4A). The population of cones which do not label with either LWS or SWSspecific antibodies are located mostly in the central retina, peaking at 78 6 30 cones/mm2 (Fig. 3D, n 5 2). Similar to that of LWS double cones, the results show a decrease in unlabeled cone densities in the centraltemporal region, adjacent to the temporal part of the LWS single cones visual streak. Unlabeled cone proportions shown in Figure 4A derive from immunohistochemistry results from both retinal wholemount and semithin plastic sections. Unlabeled cones range from 0–2.4% (n 5 3) of all cones, and represent on average 1.2% of the total cone population. There was no noticeable difference in the proportion of unlabeled cones between wholemount and section immunohistochemistry. The standard error (6 SEM) of unlabeled cone distributions remains fairly consistent irrespective of two methods being used to identify unlabeled cones. Rod densities were estimated from immunohistochemistry on horizontal plastic sections positioned

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Figure 3. Wholemount examples of the topographic distribution of cones in the left eye of the common brushtail possum. Contour lines represent regular intervals between the lowest and highest measured cell densities. Values indicate density (cells/mm2). N: nasal, V: ventral. A: Single LWS cones. B: Double LWS cones. C: SWS cones. D: Topography of unlabeled cones.

along the dorsal to ventral transect (Fig. 4B). Rod densities ranged from 199,000–288,100 cells/mm2 (n 5 1 retina), with lowest densities close to the optic disc. The retina is clearly rod-dominated. Across all samples, cones constitute 0.5 to 1.1% (n 5 1) of all photoreceptors in the dorsal to ventral transect. The highest cone percentages were associated with the region of the single LWS cones visual streak. On average, cones form 0.7% (n 5 1) of all photoreceptors along the dorsal to ventral transect: a ratio of 1 cone for every 147 rods.

Photoreceptor to ganglion cell convergence To calculate the photoreceptor to ganglion cell convergence, sampled cone and rod densities in the dorsal to ventral transect were compared to the retinal ganglion cell densities published by Freeman and Tancred (1978). The results are presented in Table 2. Rods greatly outnumber ganglion cells, with a rod-to-ganglion cell convergence ratio in excess of 400:1, particularly dorsally and ventrally, where the results show an increased density of

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rod photoreceptors. A lower rod-to-ganglion cell convergence ratio is found in the central retina. There is a close correspondence between ganglion cells and the total number of cones. The cone-to-ganglion cell ratio remains low and fairly constant in all three regions. Across most of the retina, ganglion cells outnumber cone photoreceptors. LWS single cones, responsible for the majority of the cone population, show similar convergence ratios to that of total cones, reaching a peak of two cones per ganglion cell in the dorsal retina. Double LWS cones show a low convergence both dorsally and ventrally, and a particularly low convergence ratio in the central retina, associated with the weak step function seen in the topography results (Fig. 3B). The convergence ratio is significantly below one for the entire retina for both SWS and unlabeled cones.

Microspectrophotometry Since the measuring beam was larger than most photoreceptors, only single cones with large outer

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(A1; Fig. 5A,B). Histograms of the kmax values of individual absorbance spectra used to create the mean spectra are shown in Figure 5C. All cones measured are of the LWS pigment type with a mean kmax of 544 6 4 nm (n 5 7). We were not able to measure spectral sensitivity of SWS or the unlabeled cone class, most likely due to the very low density of these cones. The average kmax of rod visual pigments is 500 6 1 nm (n 5 6).

DISCUSSION

Figure 4. A: Percentage distribution of cone subtypes 6 SEM along transects through the retinae. Dots represent percentages at individual sample locations, and the lines are thin-plate spline functions through the data points. B: Rod photoreceptor densities along a dorsal to ventral transect.

segments and groups of rods bunched together could be measured accurately. Spectral absorbance was measured from 13 photoreceptor scans which met the selection criteria outlined in the Materials and Methods. Absorbance spectra fitted well to the retinal pigment based template curve (Stavenga et al., 1993), and are assumed to contain a single chromophore type

This study has shown that the common brushtail possum Trichosurus vulpecula possesses a small population of cones in an otherwise rod-dominated retina. Nocturnal mammals typically have very few cones, ranging between 0.5–3% of all photoreceptors (Peichl, 2005). The brushtail possum fits this pattern with cone populations of 0.5–1.1% across different regions. The average ratio of 1 cone to 147 rods is the most extreme cone:rod ratio of any marsupial studied to date. The most similar marsupial species is the nocturnal South American opossum (1:130, Ahnelt et al., 1995) and northern brown bandicoot (1:120, Chase and Graydon, 1990). The brushtail possum cone:rod ratio is much smaller than those of the crepuscular honey possum (1:40, Arrese et al., 2002a), tammar wallaby (1:22, Hemmi and Gr€unert, 1999), and diurnal numbat (13:1, Arrese et al., 2000). The rod-to-ganglion cell convergence ratio is very high (range 50:1 to 436:1), improving sensitivity at low light levels, and enhancing contrast detection during scotopic vision. Even higher rod-to-ganglion cell convergence ratios have been reported in nocturnal placental mammals such as the owl monkey (60:1 to 4,400:1, Yamada et al., 2001) and the domestic cat (660:1 to 2,400:1, Steinberg et al., 1973). Although rod-dominated, the retina features three, possibly four cone subtypes: 1) a large population of LWS cones (kmax 5 544 nm), consisting of both a) single and b) double cone types. The antiserum AB5405 recognized all double cones and a subset of single cones, suggesting that their visual pigment belongs to the same LWS photoreceptor class; 2) a smaller

TABLE 2. Photoreceptor:Ganglion Cell Convergence Ratios in the Common Brushtail Possum

Rods Total Cones LWS Single LWS Double SWS Unlabeled

Dorsal(n cells/mm2)

Central(n cells/mm2)

Ventral(n cells/mm2)

436:1 (261,800) 2.5:1 (1491) 1.9: 1 (1118) 1:2.1 (287) 1:9.3 (64) 1:27 (22)

50.6:1 (202,400) 1:1.8 (2174) 1:2.1 (1906) 1:29.9 (134) 1:9.3 (64) 1:57 (70)

280:1 (279,900) 1:1.8 (1758) 1:1 (1018) 1:1.8 (554) 1:5.6 (179) 1:133 (7)

Corresponding photoreceptor densities given in parentheses. Ganglion cell densities were 600 cells/mm2 dorsally, 4000 cells/mm2 centrally, and 1000 cells/mm2 ventrally (Freeman and Tancred, 1978).

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Figure 5. Normalized mean pre-bleach (black circles) and postbleach (gray circles) absorbance spectra of visual pigments. Prebleached spectra were fitted with a Stavenga retinal pigment based template curve (solid line). A: The LWS visual pigment showing kmax of 544 6 4 nm. B: Rod visual pigments showing kmax of 500 6 1 nm. C: Histogram showing the spectral distribution of kmax values for each of the individual spectra used to create the mean spectra.

population of SWS cones; and 3) a very small population of unlabeled cones which were not recognized by either opsin-specific antibodies. The unlabeled cone population was found in both retinal wholemount and semithin plastic section immunohistochemistry. The proportion of unlabeled cones remained more consistent between methods than seen in the fat-tailed dunnart (Arrese et al., 2003; Ebeling et al., 2010). Arrese et al. (2003) originally estimated a low proportion of unlabeled cones (3%) in the dunnart, which potentially rep-

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resents an MWS cone population. The authors sequentially treated retinae with SWS and LWS-specific antibodies to analyze the topography of both cone types in the one retina, then counted MWS cone densities from the number of cones that remained unlabeled. The sequential application of antibodies may have caused Arrese et al. (2003) to label the majority of dunnart MWS cones with the LWS or SWS opsin antibody. This possibility is supported by a second dunnart study, which estimated a much higher frequency of unlabeled cones (25%) by analyzing cones in consecutive semithin sections that were alternatively labeled with SWS and LWS opsin antibodies (Ebeling et al., 2010). Double-labeling with similar SWS and LWS opsin antibodies mixed together right before application have reported no rate of failure in dichromatic mammals (Palacios et al., 2010; Schleich et al., 2010), and based on the results found in this study, seems to be a better labeling method to identify unlabeled cones than compared to treating antibodies sequentially. While the overall numbers of unlabeled cones are very low, it is possible that this irregularly spaced and sparse population of unlabeled cones represents an MWS cone population, similar to that found in a number of other marsupials (Arrese, 2002; Arrese et al., 2002b, 2003, 2005; Ebeling et al., 2010). The low density of unlabeled cones in brushtail possums, however, makes it difficult to assess how such a small photoreceptor population would contribute to visual processing. It may still be an artifact of immunolabeling, despite using two methodological techniques. Alternatively, it may be a genuine cone subpopulation, but like the double cones, may not necessarily be used during color vision. Ebeling et al. (2010) also noted a very small population of unlabeled cones present in the tammar wallaby retina (up to 4.9% dorsal-centrally). Tammar wallabies have been proven to be dichromats through multiple behavioral color vision techniques (Hemmi, 1999; Ebeling and Hemmi, 2014). Comprehensive behavioral experiments are clearly needed to test whether the common brushtail possum’s perception of colors is based on a trichromatic or dichromatic visual system. This would ultimately provide the most direct evidence for the brushtail possum’s ability to use two or three cone types to perceive and use color information. The spectral range of the brushtail possum’s visual system is similar to that of other marsupials studied to date. Based on MSP, LWS visual pigments have peak sensitivity (kmax) of 544 nm. Other marsupials range from 535 nm to 562 nm (Hemmi et al., 2000; Arrese et al., 2002b, 2005; Deeb et al., 2003; Hunt et al., 2009b; Jacobs and Williams, 2010; Palacios et al., 2010). Deeb (2010) reported an LWS kmax of 556 nm

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in the brushtail possum based on unpublished data. It is not clear, however, whether the unpublished study used MSP, amino-acid sequencing, or both in the analysis. It is difficult to explain this difference between the LWS visual pigment values reported in this study and that reported by Deeb (2010) without a direct comparison of the methodology available. Both MSP and electroretinography (ERG) measurements in marsupials, however, have also previously been reported to differ from the kmax values determined by amino-acid sequencing. Arrese et al. (2002b) reported a kmax of 350 nm for the UV visual pigment when using MSP, while amino-acid sequencing predicted a kmax of 360 nm (Strachan et al., 2004). In the tammar wallaby, ERG revealed an LWS kmax of 539 nm (Hemmi et al., 2000), but amino-acid sequencing determined an LWS kmax of 530 nm (Deeb et al., 2003). While our attempts to measure an SWS visual pigment with MSP were unsuccessful, Deeb (2010) also reported that, based on unpublished data, the brushtail possum has an SWS pigment sensitive to ultraviolet light (kmax of 360 nm). Several marsupial species have also been reported to have ultraviolet-sensitive visual pigments (Arrese et al., 2002b, 2005; Cowing et al., 2008; Hunt et al., 2009b; Palacios et al., 2010). Only the tammar wallaby and quokka have been reported to possess a violet/blue visual pigment (Hemmi, 1999; Deeb et al., 2003; Arrese et al., 2005). Whether the shift of the SWS visual pigment towards longer wavelengths is a common feature among only the macropod marsupials remains to be seen. MSP also found that the peak spectral sensitivities (kmax) of rod visual pigments in the brushtail possum was 500 nm, which is typical for most terrestrial mammals (Hunt et al., 2009a).

Cone topography and visual ecology There are substantial differences in the density and distribution of photoreceptors in the brushtail possum retina, supporting the theory that retinal topography is caused by ecological factors. The topographies of different cone types vary markedly from each other. Single LWS cones form a weak horizontal streak in the central retina, and closely resemble that of the ganglion cell distribution (Freeman and Tancred, 1978). A visual streak has previously been suggested to correlate with open habitats, providing increased visual resolution and movement sensitivity along an extended flat terrain (Hughes, 1975, 1977). The distribution of ganglion cells in the arboreal common brushtail possum initially led Freeman and Tancred (1978) to conclude that the "terrain" hypothesis of retinal topographic organization is false. The brushtail possum, however, is not exclusively arboreal (Hughes, 1981). Brushtail possums commonly

use ground dens during cooler temperatures or when trees are scarce (Kerle, 1984; Green and Coleman, 1987; Cowan, 1989). They are generalist and opportunistic feeders, known to occasionally forage on the ground (Hughes, 1981; MacLennan, 1984; Nugent et al., 2001). Brushtail possums are also susceptible to native ground predators (Newsome et al., 1983; Slip and Shine, 1988). A greater range of high visual acuity across the horizon will be advantageous when ground foraging, or detecting ground predators while they are moving between trees, and are most vulnerable. The visual streak is also quite weak when compared to the ganglion cell distributions in exclusively terrestrial marsupials, such as the red kangaroo (Hughes, 1975). Specialized regions in the ventral retina suggest that visual information in the upper visual field is particularly important for common brushtail possums. Double LWS cones are densely located ventrally, and also form a "negative" visual streak in the same retinal location as the single LWS cones visual streak. This creates a weak step function from the dorsal to ventral retina. Considering the strong regionalization of single and double LWS cones, similar to the topographical variation typically seen between LWS and SWS cone classes, it seems likely that single and double cones serve different visual functions. Whether their function involves chromatic (Neumeyer, 1986; Hughes et al., 1998; Pignatelli et al., 2010) or achromatic tasks such as improved luminance, polarization, or movement detection (Young and Martin, 1984; Cameron and Pugh, 1991; Novales Flamarique et al., 1998; Vorobyev and Osorio, 1998), double cones specifically sample the upper visual field, and not the visual horizon. The regional specialization of double cones in the ventral retina appears to be a common feature among the marsupials. A ventral or peripheral increase of double cones has been noted in several marsupial species (Hemmi and Gr€unert, 1999; Arrese et al., 2003; Palacios et al., 2010), and low double cone densities in the single cone visual streak has also been described topographically in the South American opossum Didelphis marsupialis (Ahnelt et al., 1995). The unlabeled cone population also appears to not be associated with the visual horizon, although, as mentioned previously, their sparsity in the retina makes it difficult to assess their functionality. Alongside MWS cones, the visual function of double cones in marsupial vision certainly warrants further investigation. Common brushtail possums are the first Australian species known with a higher ventral concentration of SWS cones. The honey possum has a uniform SWS distribution, and all other species have greater SWS densities in the dorsal retina (Hemmi and Gr€unert, 1999;

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Arrese et al., 2003, 2005). The only other species with a ventral increase of SWS cones is the South American marsupial Thylamys elegans, which has a visual streak of SWS cones through the nasal-temporal retina (Palacios et al., 2010). An increased density of SWS cones in the ventral retina has been suggested to be an adaptation to natural skylight (Szel et al., 1994; Szel et al., 1996). The upper visual field for an arboreal common brushtail possum would view dense tree foliage against skylight. It is possible that brushtail possums have SWS cones in the ventral retina to improve both color vision and contrast against the moving foliage. This would be particularly advantageous for predator avoidance, as large arboreal predators such as the powerful owl Ninox strenua (Cooke et al., 2006) would appear in the upper visual field. Whether the LWS double cones could serve the same function in relation to the brushtail possum’s visual ecology remains to be seen. Cones-to-ganglion cell convergence ratios remain fairly constant across the brushtail possum retina (1:2 to 2.5:1). This cone-to-ganglion cell ratio is similar to that in the North American opossum, which has a constant convergence of 3:1 across the retina (Kolb and Wang, 1985). The honey possum has a higher proportion of cones in the central and mid-ventral retina alongside a high ganglion cell density, and hence a higher convergence ratio (12:1, Arrese et al., 2002a). In most mammalian retinae, cone-to-ganglion cell convergence ratios tend to be low in the central retina (domestic cat 4:1, California ground squirrel 1.8:1, sheep 9:1; Steinberg et al., 1973; Long and Fisher, 1983; Shinozaki et al., 2010), and increase towards the periphery (domestic cat 19:1, California ground squirrel 5:1, sheep 15:1). Given that brushtail possums are predominantly nocturnal, we hypothesize that ganglion cell densities are optimized for scotopic vision, and that they have minimized the number of cones necessary to serve the ganglion cells for photopic vision, at the cost of sensitivity. This would mean that they may have no loss of acuity when they go from day to night. Behavioral measures of visual acuity and contrast sensitivity during various light levels would be required to test this hypothesis.

CONCLUSION In this study we describe the presence and distribution of photoreceptors in the common brushtail possum. Photoreceptor classes and morphological types show strong regional specializations across the retina, particularly in the horizontal and upper visual field. A small number of unlabeled cones present alongside SWS and LWS cones may form a third cone opsin population. It is difficult to assess, however, how such a

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small population of cones contribute to visual processing without behavioral evidence of trichromacy.

ACKNOWLEDGMENTS The authors thank W. Ebeling, S.T.D. New, and R. Albarracin for help and advice with the immunohistochemistry. We are grateful for the JH455 antibody provided by U. Gr€unert (University of Sydney, Australia), originally donated by J. Nathans (Johns Hopkins University School of Medicine, Baltimore, MD). Comments from J. Zeil and J.P. Coimbra on earlier versions of the article is greatly appreciated.

CONFLICT OF INTEREST The authors declare that they have no competing interests.

ROLE OF AUTHORS Study concept and design: LMV, JMH, KV. Collection and acquisition of data: LMV, BK. Analysis and interpretation of data: LMV, JMH. Drafting of the article: LMV. Critical revision of the article for important intellectual content: LMV, JMH, KVK, BK. All authors have read and approved the final article.

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